Regulation of mt1-mmp activity

ABSTRACT

It has been considered that an activation of proMMP-2 induced by MT1-MMP is one of the important steps for invasion of tumor cells such as cancers, but its detailed mechanism for an expression of activation has been unknown. Thus, it has been required that methods for preventing/treating various diseases are developed by elucidating the mechanism of activation expression of proMMP-2 induced by MT1-MMP and elucidating various actions potentially involving MT1-MMP in addition to invasion and metastasis of cancers. A formation of a complex formed of multiple MT1-MMP, particularly a homodimer formation of MT1-MMP is crucial for the activation of proMMP-2 induced by MT1-MMP, and the homodimer formation is based on functions of the PEX domain of MT1-MMP. Utilizing such findings, the homodimer formation of MT1-MMP is inhibited and the activation of proMMP-2 is inhibited leading to developing methods to control invasion/metastasis of cancers.

FIELD OF THE INVENTION

[0001] The present invention relates to substances which inhibit theformation of complexes comprised of a plurality of membrane-type 1matrix metalloproteinases (MT1-MMPs). Particularly, the presentinvention relates to substances which inhibit the homophilic dimerformation of MT1-MMP present on a cell surface.

[0002] The present invention relates to methods for controlling variousphysiological and biological processes attributable to the activation ofproMMP-2, which comprise inhibiting the homophilic dimer formation ofMT1-MMP thereby inhibiting the activation of proMMP-2 and topharmaceutical drugs therefor. The present invention further providespharmaceuticals and therapeutic methods for the inhibition of cancerinvasion and metastasis based on regulation mechanisms of MT1-MMPactivity via the hemopexin-like (PEX) domain of MT1-MMP. Morespecifically, the present invention relates to MT1-MMP complex formationinhibitors, proMMP-2 activation inhibitors, blockers for cell migration,invasion and/or metastasis, associated with the homophilic dimerformation of MT1-MMP. Further, the present invention relates to methodsfor inhibiting the homophilic dimer formation of MT1-MMP and relatedmethods, to substances (such as MT1-PEX) having an inhibitory activityon the homophilic dimer formation of MT1-MMP present on a cell surface,to nucleic acids coding for the same and uses of said nucleic acids. Thepresent invention also relates to antibodies against the PEX protein ofMT1-MMP or fragments thereof or polypeptides having substantiallyequivalent activity as compared to the same and uses thereof. Thepresent invention relates to screening methods for inhibitory substancesagainst the homophilic dimer formation of MT1-MMP and reagents therefor.

BACKGROUND OF THE INVENTION

[0003] Matrix metalloproteinases (MMPs) are of the zinc-dependentendopeptidase family which degrade various constitutive proteins atextracellular matrices (ECM) and basement membrane components. Theseenzymes are involved in rearrangement of connective tissues such asnormal embryonic development, bone growth or wound healing and the like(Woessner, J. F., FASEB J., 5:2145-2154, 1991; Matrisian, L. M.,BioEssays, 14:455-463, 1992; Birkedal-Hansen, H., Moore, W. G. I.,Bodden, M. K., Windsor, L. J., Birkedal-Hansen, B., DeCarlo, A., andEngler, J. A., Crit. Rev. Oral Biol. Med., 4:197-250, 1993). It hasbecome known that these are also involved in various processes ofdiseases such as atherosclerosis (Henney, A. M., Wakeley, P. R., Davies,M. J., Foster, K., Hembry, R., Murphy, G., and Humphries, S., Proc.Natl. Acad. Sci. USA, 88:8154-8158, 1991), pulmonary emphysema(Hautamaki, R. D., Kobayashi, D. K., Senior, R. M., and Shapiro, S. D.,Science, 277:2002-2004, 1997), rheumatoid arthritis (Murphy, G., andHembry, R. M., J. Rheumatol., 19:61-64, 1992), and invasion/metastasisof cancers (Stetler-Stevenson, W. G., Aznavoorian, S., and Liotta, L.A., Annu. Rev. Cell Biol., 9:541-573, 1993).

[0004] To the present, MMPs (MMP-1 (collagenase)); MMP-2 (gelatinase A);MMP-3 (stromelysin-1); MMP-7 (matrilysin); MMP-8 (neutrophilcollagenase); MMP-9 (gelatinase B); MMP-10 (stromelysin-2); MMP-11(stromelysin-3); MMP-12 (macrophage elastase); MMP-13 (collagenase-3);MMP-14 (MT1-MMP); MMP-15 (MT2-MMP); MMP-16 (MT3-MMP); MMP-17 (MT4-MMP);MMP-18 (collagenase-4); MMP-19; MMP-20 (enamelysin); MT5-MMP and thelike) analyzed at an amino acid level have been known (Woessner, J. F.,FASEB J., 5:2145-2154, 1991; Matrisian, L. M., BioEssays, 14:455-463,1992; Birkedal-Hansen, H., Moore, W. G. I., Bodden, M. K., Windsor, L.J., Birkedal-Hansen, B., DeCarlo, A., and Engler, J. A., Crit. Rev. OralBiol. Med., 4:197-250, 1993; Gururajan, R., Grenet, J., Lahti, J. M.,and Kidd, V. J., Genomics, 52:101-106, 1998; Velasco, G., Pendas, A. M.,Fueyo, A., Knauper, V., Murphy, G., and Lopez-Otin, C., J. Biol. Chem.,274: 4570-4576, 1999). These MMPs are classified into at least 4 typesof subfamilies: collagenases, gelatinases, stromelysins andmembrane-type MMPs (MT-MMPs) depending on their primary structures,substrate specificity and cellular distribution, and also can be broadlysubgrouped into soluble MMPs and membrane-anchored MMPs (membrane-typeMMPs, MT-MMPs). Soluble MMPs are believed to be related to ECMdegradation in broad areas of tissues. On the other hand, MT-MMPs arebelieved to be related to ECM degradation surrounding cells because theyare connected to the plasma membrane (Seiki, M., Apmis, 107:137-143,1999). MMPs are composed of preserved domain structures of apre/propeptide, a catalytic domain, a hinge, and a hemopexin-like domain(Nagase, H., et al., J. Biol. Chem., 274: 21491-21494, 1999).

[0005] MT-MMPs subfamilies have been most recently reported as asubclass of MMPs, and five types of members (MT1-MMP, MT2-MMP, MT3-MMP,MT4-MMP and MT5-MMP) have been isolated and identified by degeneratedprimers for the preserved domains of MMPs and RT-PCR (Sato, H., Takino,T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M.,Nature, 370:61-65, 1994; Will, H., and Hinzmann, B., Eur. J. Biochem.,231: 602-608, 1995; Takino, T., Sato, H., Shinagawa, A., and Seiki, M.,J. Biol. Chem., 270:23013-23020, 1995; Puente, X. S., Pendas, A. M.,Llano, E., Velasco, G., and Lopez-Otin, C., Cancer Res., 56:944-949,1996; Japanese Unexamined Patent Publication No. 2000-270874; Pei, D.,J. Biol. Chem., 274:8925-8932, 1999; Kajita, M. et al., FEBS Lett., 457:353-356, 1999).

[0006] MT-MMPs are type I membrane proteins which have a singletransmembrane domain and a short cytoplasmic tail following thehemopexin domain characteristics of many MMPs. Additionally, aninsertion of basic amino acids is commonly present between thepropeptide and an active domain. Cleavage by a furin or furin-likeenzyme induces the activation of these membrane proteins (Pei, D., andWeiss, S. J., J. Biol. Chem., 271:9135-9140, 1996; Sato H., Kinoshita,T., Takino, T., Nakayama, K., and Seiki, M., FEBS Lett., 393:101-104,1996; Cao, J., Rehemtulla, A., Bahou, W., and Zucker, S., J. Biol.Chem., 271:30174-30180, 1996).

[0007] When cells migrate, invade and metastasize in the tissue, thedegradation of extracellular matrices surrounding them is an essentialstep. Those playing a pivotal role in the step are the enzyme groupcalled MMPs. Among others, MT1-MMP expressed on the surface of a cellmembrane is given importance in its roles in migration, invasion,metastasis and angiogenesis of cancers.

[0008] MT1-MMP is the enzyme also called membrane-type 1 matrixmetalloproteinase (MT1-MMP) or MMP-14 (MEROPS ID: M10,014), and onereported as the product of a gene occupying a chromosomal locus,14q11-q12 in humans (Migon, C. et al., Genomics, 28:360-361, 1995) ofwhich a detailed structure and profiles have been demonstrated bysuccessful DNA cloning and recombinant protein expression thereof (Sato,H. et al., Nature, 370:61-65, 1994; Takino, T., et al., Gene,155:293-298, 1995; Japanese Unexamined Patent Publication Nos.Hei-7-203961, Hei-7-303482; GenBank™ accession number: D26512). Thepresence of MT1-MMP has been detected in the dogs, goats, rabbits, wildboars, mice, etc., in addition to humans. cDNA of human MT1-MMP encodes582 amino acid residues (EMBL accession No. D26512, E09720 and E10297;SWISS-PROT: P50281), of which the structure is composed of a signalpeptide followed by a propeptide domain, an insertion sequence composedof 10 specific amino acid residues similar to stromelysin-3 (a potentialsequence of a furin-like enzyme recognition site), a core enzyme domainhaving a potential site with a zinc binding site, a hinge domain, and ahemopexin-like domain encompassing a transmembrane domain.

[0009] Thus, MT1-MMP has been thought to have a transmembrane (TM)domain at the C terminus and to exert its biological activity on thecell membrane. To the present, it has been found that MT1-MMP activatesa progelatinase A (proMMP-2) which is also a member of the same familyand a basement membrane degrading enzyme, on the cell membrane, andfurther that MT1-MMP per se degrades various ECM molecules such ascollagen type I, II and III, fibronectin, laminin, vitronectin andaggrecan. Also, MT1-MMP has been shown to promote the processes of tumorinvasion and metastasis (Seiki, M., Apmis, 107:137-143, 1999; Sato, H.,et al., Nature, 370:61-65, 1994). MT1-MMP also activates the other MMPssuch as proMMP-2 (Sato, H., et al., Nature, 370:61-65, 1994) andprocollagenase-3 (proMMP-13) (Knauper, V., et al., J. Biol. Chem.,271:17124-17131, 1996).

[0010] Thus, the expression of MT1-MMP is thought to initiate multipleproteinase cascades on a cell surface. And it has been shown thatMT1-MMP is involved in not only invasion and metastasis of cancer cells(Seiki, M., Apmis, 107:137-143, 1999; Sato, H., et al., Nature,370:61-65, 1994), but also other physiological processes such asangiogenesis (Hiraoka, N., et al., Cell, 95:365-377, 1998; Zhou, Z., etal., Proc. Natl. Acad. Sci. USA, 97:4052-4057, 2000) and skeletaldevelopment (Zhou, Z., et al., Proc. Natl. Acad. Sci. USA, 97:4052-4057,2000; Holmbeck, K., et al., Cell, 99:81-92, 1999).

[0011] Thus, MT1-MMP is considered to be a tool required forphysiological and pathological cellular invasion in tissue. Therefore,it is important to elucidate action mechanisms of MT1-MMP on a cellsurface.

[0012] MT1-MMP has been shown to promote invasion of cancer cells byactivating proMMP-2 and by directly degrading extracellular matrices.The activation of proMMP-2 on a cell surface by MT1-MMP appears to be animportant step in tumor invasion. This is because MMP-2 degrades type IVcollagen and laminin which are major components of the basement membrane(Stetler-Stevenson, W. G. et al., Annu. Rev. Cell Biol., 9:541-573,1993). The occurrence of active MMP-2 is also significantly correlatedwith tumor invasion (Tokuraku, M. et al., Int. J. Cancer, 64:355-359,1995; Nomura, H. et al., Cancer Res., 55:3263-3266, 1995). Itsactivation step includes the tri-molecular complex formation of MT1-MMP,TIMP-2 and proMMP-2 (Strongin, A. Y., et al., J. Biol. Chem.,270:5331-5338, 1995). However, the activity expression mechanisms ofMT1-MMP on a cell membrane have not been ascertained in detail.Therefore, it has been unknown what should be done to strictly controlthe activity of MT1-MMP. Thus, there have been problems in that measuresto control migration, invasion and metastasis of cells such as cancerscannot be demonstrated.

SUMMARY OF THE INVENTION

[0013] The present inventors have conducted an intensive study touncover the elicitation mechanism of MT1-MMP activity. As a result, theinventors have successfully found that MT1-MMP forms a homo-dimercomplex through the hemopexin-like (PEX) domain, and that the complexformation is essential for the activation of proMMP-2 on the cellsurface. Furthermore, it has been also found that the expression of PEXwithout a catalytic site of MT1-MMP on a cell surface directly preventsan association of two enzymes and inhibits the activation of proMMP-2 ina dose-dependent manner. In addition, it has been found that theexpression of PEX without the catalytic site of MT1-MMP suppressesinvasion and metastasis of tumor cells. As a result, the inventors havecompleted the present invention.

[0014] The present invention provides:

[0015] (1) A membrane-type 1 matrix metalloproteinase (MT1-MMP) complexformation inhibitor which inhibits the formation of a complex comprisedof plural MT1-MMPs.

[0016] (2) A proMMP-2 activation inhibitor which inhibits the formationof a complex comprised of plural MT1-MMPs.

[0017] (3) A blocker for cell migration, invasion and/or metastasiswhich inhibits the activation of proMMP-2 through inhibiting theformation of a complex comprised of plural MT1-MMPs.

[0018] (4) The blocker according to the above (3), wherein the cell is acancer cell.

[0019] (5) The inhibitor or blocker according to any of the above (1)through (4), wherein the complex comprised of plural MT1-MMPs is anMT1-MMP homophilic dimer.

[0020] (6) The inhibitor or blocker according to any of the above (1)through (5), wherein the complex formation is executed at least withMT1-MMP existing on a cell surface.

[0021] (7) A method for inhibiting MT1-MMP complex formation whichcomprises inhibiting the formation of a complex comprised of pluralMT1-MMPs.

[0022] (8) A method for inhibiting the activation of proMMP-2 whichcomprises inhibiting the formation of a complex comprised of pluralMT1-MMPs.

[0023] (9) A method for blocking cell migration, invasion and/ormetastasis which comprises inhibiting the activation of proMMP-2 throughinhibiting the formation of a complex comprised of plural MT1-MMPs.

[0024] (10) The method according to the above (9), wherein the cell is acancer cell.

[0025] (11) The method according to any of the above (7) through (10),wherein the complex comprised of plural MT1-MMPs is an MT1-MMPhomophilic dimer.

[0026] (12) The method according to any of the above (7) through (11),wherein the complex formation is executed at least with MT1-MMP existingon a cell surface.

[0027] (13) A substance which is active in inhibiting the homophilicdimer formation of MT1-MMP that exists on a cell surface.

[0028] (14) The substance according to the above (13) which is anMT1-MMP hemopexin-like domain (PEX) protein or a fragment thereof, or asubstance which has substantially equivalent activity as compared to thesame.

[0029] (15) The substance according to the above (14), wherein thesubstance is from Cys³¹⁹ to Gly⁵³⁵ of Met plus MT1-MMP, or a proteinhaving substantially equivalent activity as compared to the same.

[0030] (16) A nucleic acid selected from the group consisting of:

[0031] (i) a nucleic acid coding for MT1-MMP PEX;

[0032] (ii) a nucleic acid coding for a fragment of MT1-MMP PEX;

[0033] (iii) a nucleic acid hybridizable to the nucleic acid as setforth in the above (i); and

[0034] (iv) a nucleic acid having substantially equivalent activity ascompared to the nucleic acid as set forth in any of the above (i)through (iii).

[0035] (17) The nucleic acid according to the above (16), wherein thenucleic acid is a chimeric molecule in which the transmembrane(TM)/cytoplasmic (CP) domains of MT1-MMP are substituted with nervegrowth factor receptor (NGFR) TM/CP, or a molecule having substantiallyequivalent activity as compared to the same.

[0036] (18) The nucleic acid according to the above (16), which is anucleic acid coding for from Cys³¹⁹ to Gly⁵³⁵ of Met plus MT1-MMP, or anucleic acid having substantially equivalent activity as compared to thesame.

[0037] (19) A vector comprising a nucleic acid according to any one ofthe above (16) through (18).

[0038] (20) A transformed cell comprising (i) a nucleic acid accordingto any of the above (16) through (18) or

[0039] (ii) a vector according to the above (18).

[0040] (21) The transformed cell according to the above (20) whichexpresses a chimeric molecule in which MT1-MMP TM/CP is substituted withNGFR TM/CP, or a molecule having substantially equivalent activity ascompared to the same.

[0041] (22) A gene therapeutic agent which comprises (i) a nucleic acidaccording to any one of the above (16) or

[0042] (18) or (ii) a vector according to the above. (19).

[0043] (23) An antibody against (i) an MT1-MMP PEX protein or a fragmentthereof, or (ii) a polypeptide having substantially equivalent activityas compared to the same.

[0044] (24) A substance capable of binding to MT1-MMP PEX.

[0045] (25) The substance according to the above (22) which has MT1PEXactivity or substantially equivalent activity as compared to MT1PEX.

[0046] (26) A pharmaceutical or veterinary drug comprising the inhibitoror blocker according to any of the above (1) through (6), or thesubstance, nucleic acid, vector, transformed cell, agent or antibodyaccording to any of the above (13) through (16), (18) through (20), and(22) through (25).

[0047] (27) The pharmaceutical or veterinary drug according to the above(26) for suppressing and/or blocking cell migration, invasion and/ormetastasis.

[0048] (28) The pharmaceutical or veterinary drug according to the above(26), wherein the cell is a cancer cell.

[0049] (29) A screening which comprises screening with using, as amarker, the formation of a complex comprised of plural MT1-MMPs for asubstance which inhibits the formation of said complex comprised of saidplural MT1-MMPs.

[0050] (30) The screening according to the above (29), wherein thecomplex comprised of the plural MT1-MMPs is an MT1-MMP homophilic dimer.

[0051] (31) The screening according to the above (29) or (30), wherein achimeric molecule is used in which MT1-MMP transmembrane(TM)/cytoplasmic (CP) domains are substituted with nerve growth factorreceptor (NGFR) TM/CP.

[0052] (32) A substance which (i) inhibits the formation of a complexcomprised of plural MT1-MMPs and (ii) is obtained by the screeningaccording to any of the above (29) through (31).

[0053] The above objectives and other objectives, features, advantages,and aspects of the present invention are readily apparent to thoseskilled in the art from the following disclosures. It should beunderstood, however, that the description of the specification includingthe following best modes of carrying out the invention, examples, etc.is illustrating preferred embodiments of the present invention and givenonly for explanation thereof. It will become apparent to the skilled inthe art that a great number of variations and/or alterations (ormodifications) of this invention may be made based on knowledge from thedisclosure in the following parts and other parts of the specificationwithout departing from the spirit and scope thereof as disclosed herein.All of the patent publications and reference documents cited herein forillustrative purposes are hereby incorporated by reference into thepresent disclosure.

[0054] As used herein, the term “and/or” means that both (1) combinedconjunction and (2) selective conjunction are present. For instance, inthe case of “invasion and/or metastasis”, it is used to mean that both(1) “invasion and metastasis” and (2) “invasion or metastasis” areincluded. In other cases, the term “and/or” is used to mean that both(1) combined conjunction and (2) selective conjunction are included inthe same way.

BRIEF DESCRIPTION OF THE DRAWINGS

[0055]FIG. 1: A schematic illustration of MT1-MMP mutant constructs asused herein.

[0056]FIG. 2: Tests for the activation of proMMP-2 by MT1-F on a cellsurface. COS1 cells were transfected with expression vectors for MT1-MMPmutants; MT1-F or MT1Cat-F, or a vector alone (Mock), and then culturedin a serum-free culture medium in the presence of purified proMMP-2(0.25 μg/ml) at 37° C. for 18 hrs. The resultant cell lysates wereexamined for the expression of mutants, and the culture supernatantswere examined for the activation of proMMP-2. The upper panel indicatesthe Western blotting analysis with monoclonal anti-FLAG M2 antibody forcell lysates obtained from the transfected cells. The lower panelindicates the gelatin zymographic patterns for culture media.

[0057]FIG. 3: A schematic illustration of MT1EAΔ TM expressed in E.coli. E/A indicates that Glu²⁴⁰ is mutated with Ala.

[0058]FIG. 4: Tests for a homophilic complex formation by the ectodomainof MT1-MMP. The gel permeation analysis profiles of MT1EA Δ TM at threedifferent concentrations. The inset shows the SDS-PAGE analysis ofMTLEAΔ TM under reducing and non-reducing conditions.

[0059]FIG. 5: Tests for the formation of the homophilic complex of theectodomain of MT1-MMP. The products obtained by crosslinking MT1EAΔ TMwith a chemical coupler, DSG, were examined by SDS-PAGE analysis.

[0060]FIG. 6: In regard to the formation of a homodimer complex ofMT1-MMP PEX, MT1EA Δ TM(50 μg/ml) was treated with trypsin, leading to acleavage between a catalytic domain and PEX, and then the samples wereapplied on a Superdex 75 gel permeation column. The results are shown inthis drawing. The inset indicates purified PEX and catalytic domain(Cat) positions on SDS-PAGE analysis under reducing and non-reducingconditions.

[0061]FIG. 7: Superdex 75 gel permeation column profiles of MT1-MMP PEXand mouse MT4-MMP PEX in regard to the homodimer complex formation ofMT1-MMP PEX. The inset indicates mouse MT4-MMP PEX on SDS-PAGE analysisunder reducing and non-reducing conditions.

[0062]FIG. 8: A schematic illustration of various mutant MT1-MMPsconstructed.

[0063]FIG. 9: Tests for a dimer formation by the ectodomain of MT1-MMPon the cell surface. COS1 cells were transfected with the expressionvectors for MT1-F/NGFR, MT1PEX-F/NGFR, MT1Cat-F/NGFR or MT4PEX-F/NGFR,or a vector alone (Mock). The cell lysates were analyzed by Westernblotting using a monoclonal anti-FLAG M2 antibody (upper panel:Anti-FLAG) and an anti-phosphotyrosine PY20 monoclonal antibody (lowerpanel: Anti-PY).

[0064]FIG. 10: Tests for a dimer formation by the ectodomain of MT1-MMPon the cell surface. COS1 cells were co-transfected with vectors forMT1-F/NGFR and MT1PEX-F at the indicated DNA ratio. The cell lysateswere analyzed by Western blotting using anti-FLAG M2 (Anti-FLAG) andanti-phosphotyrosine PY20 (Anti-PY). The relative intensity of the bandsdetected with PY20 is shown.

[0065]FIG. 11: The efficacy of MT1PEX-F on the proMMP-2 activation byMT1-F on the cell surface. COS1 cells were co-transfected with vectorsfor MT1-F and MT1PEX-F at the indicated DNA ratio. Then, the above cellswere reacted with purified proMMP-2 (0.25 μg/ml) in a serum-free mediumat 37° C. for 18 hrs. MMP-2 in the culture supernatants was analyzed bygelatin zymography (upper panel), and the cell lysates were analyzed byWestern blotting using a monoclonal anti-FLAG M2 antibody (lower panel).

[0066]FIG. 12: The efficacies of MT1PEX-F on the proteolytic activityand TIMP-2 binding ability of MT1-F expressed on a cell surface areshown. As is shown in FIG. 12 through FIG. 14, CHO-K1 cells weretransfected with the expression vectors for MT1-F, MT1PEX-F or MT1Cat-F,or a vector alone (Mock). The cells were then treated with trypsin, andseeded on DQ™-gelatin coated cover slips for proteolytic activity tests(FIG. 13) and in 24-well plates for TIMP-2 binding assays (FIG. 12) andfor proMMP-2 activation (FIG. 14). Results are all from the sametransfection experiment. This figure indicates the following TIMP-2binding assay: The transfected cells in the 24-well plates wereincubated with 25 nM of ¹²⁵I-labeled TIMP-2 in the presence or absenceof BB-94 (50 μM) at 0° C. for one hour. The MT1-MMP specific binding wasobtained by subtracting the data in the presence of BB-94 (50 μM) fromeach experiment.

[0067]FIG. 13: As in the case with FIG. 12, the effects of MT1PEX-F onproteolytic activity and TIMP-2 binding ability of MT1-F expressed onthe cell surface are shown. The transfected cells on DQ™-gelatin coatedcover slips were incubated in the medium at 37° C. for 18 hrs. Generatedfluorescence was analyzed by confocal microscopy.

[0068]FIG. 14: As in the case with FIG. 12, the efficacies of MT1PEX-Fon the proteolytic activity and TIMP-2 binding ability of MT1-Fexpressed on a cell surface are shown. The same transfected cells in the24-well plates were reacted with purified proMMP-2 in the serum-freemedium at 37° C. for 18 hrs. The cells and culture supernatants wereanalyzed by Western blotting using a monoclonal anti-FLAG M2 antibody(upper panel) and by gelatin zymography (lower panel), respectively.

[0069]FIG. 15: The efficacy of MT1PEX-F on the matrigel invasive actionof HT1080 cell is shown. HT1080 cells were co-transfected with thevectors for MT1PEX-F and GFP or transfected with the expression vectorfor GFP alone. Then, the cells were subjected to matrigel invasiveanalysis by the modified Boiden-chamber method as described in“Examples” herein. TIMP-2 at 0.5 μg/ml was added to an upper and lowerchambers. GFP-positive cells on the surface of the lower chamber werecounted using fluorescent microscopy. The numbers of GFP-positive cellsamong Mock and MT1PEX-F transfected cells were 1.31×10⁴ and1.12×10⁴/1×10⁵, respectively.

[0070]FIG. 16: A schematic illustration of MT1-MMP mutants as used for ahomophilic complex formation by an interaction with the PEX domain ofMT1-MMP. Each symbol indicates as follows: Pro: propeptide; FLAG: Flagepitope; Myc: c-Myc epitope; CD: catalytic domain; H: hinge domain; PEX:hemopexin-like domain; and TM: transmembrane domain.

[0071]FIG. 17: As in the case with FIG. 16, COS1 cells were transfectedwith the vectors for MT1-F and/or MT1-Myc. The cells were lysed, andsubjected to immunoprecipitation using anti-FLAG. M2 antibody-conjugatedbeads. Whole cell lysates (Whole Cell) and immunoprecipitates(Anti-FLAGIP) were analyzed by Western blotting using an anti-FLAG M2antibody (Anti-FLAG) or an anti-c-Myc antibody (Anti-Myc).

[0072]FIG. 18: As in the case with FIG. 16, the expression plasmids forMT1-F, MT1Cat and/or MT1PEX were transfected into COS1 cells. The cellswere lysed, and subjected to immunoprecipitation using anti-FLAG M2antibody-conjugated beads as in the case with FIG. 17. The samples wereanalyzed by Western blotting using an anti-FLAG M2 antibody (Anti-FLAG),anti-MT1-MMP PEX (Anti-PEX) or anti-MT1Cat (Anti-Cat).

[0073]FIG. 19: A schematic illustration of MT1-MT4PEX as used for thestudy of MT1-MT4PEX on the proMMP-2 activation on the cell surface. ThePEX domain of MT4-PEX is derived from MT4-MMP.

[0074]FIG. 20: As in the case with FIG. 19, the activation of proMMP-2by MT1-MMP is shown. COS1 cells were transfected with the expressionplasmids for MT1-MMP, MT1-MT4PEX or a vector alone (Mock), and reactedwith proMMP-2 in the serum-free medium. The culture media were analyzedfor proMMP-2 processing by zymography (upper panel). The cell lysateswere analyzed by Western blotting using an anti-MT1Cat (mid panel). Thetransfected cells were subjected to surface biotinylation, and analyzedby Western blotting using an anti-MT1Cat (lower panel, SurfaceBiotinylation).

[0075]FIG. 21: As in the case with FIG. 19, the binding of proMMP-2 tothe MT1-MT4PEX-expressing cells was examined. The samples were analyzedby gelatin zymography.

[0076]FIG. 22: As in the case with FIG. 19, the MT1-MT4PEX-expressingcells were cultured on DQ gelatin coated cover slips and then examinedfor their in situ gelatin degrading activity. The gelatin degradingactivity was visualized as fluorescent areas. The bright-field image isalso shown. Bar: 50 μm.

[0077]FIG. 23: Tests for the dimer formation by the ectodomain ofMT1-MMP on the cell surface. COS1 cells were transfected with vectorsfor MT1-F/NGFR and/or MT1PEX-F, MT1Cat-F or MT1-F. The cells weresubjected to Western blotting using anti-FLAG M2 (Anti-FLAG) oranti-PY20 (Anti-PY). The relative intensity of the bands detected byPY20 was shown by analyzing with an NIH image.

[0078]FIG. 24: In regard to the action of constitutively active Rac1(Rac1DA) and the potential activation of proMMP-2 on the dimer formationof MT1-MMP, the efficacy of Rac1DA on the dimer formation of MT1-F/NGFRis shown. COS1 cells were transfected with the expression plasmids forMT1-F/NGFR, MT1PEX-F and/or Rac1DA. The cell lysates were subjected toWestern blotting using PY20 (Anti-PY) and an anti-FLAG M2 antibody(Anti-FLAG). The relative intensity of the bands detected by PY20 wasshown by analyzing with NIH image.

[0079]FIG. 25: As in the case with FIG. 24, the transfected COS1 cellswere stained with PY20 (Anti-PY). F-actin was also stained withAlexa488-conjugated phalloidin (Alexa488 phalloidin). A white arrowindicates a site where F-actin and a PY signal were co-localized. Bar:10 μm.

[0080]FIG. 26: As in the case with FIG. 24, COS1 cells were transfectedwith the expression plasmids for MT1-MMP and Rac1DA, and reacted withpurified proMMP-2. The culture media were analyzed for proMMP-2processing by zymography (upper panel). The cell lysates were analyzedby Western blotting using an anti-MT1PEX antibody (lower panel).Relative activation of proMMP-2 to its active form was estimated bymeasuring the intensity of proMMP-2 bands with an NIH image.

[0081]FIG. 27: As in the case with FIG. 24, the transfected COS1 cellswere stained with an anti-FLAG M1 antibody. F-actin was also stainedwith Alexa488-conjugated phalloidin (Alexa488 phalloidin). A white arrowindicates a site where F-actin and a FLAG signal were co-localized. Bar:10 μm.

[0082]FIG. 28: In regard to the efficacy of MT1PEX-F on the proMMP-2activation by MT1-MMP on the cell surface, COS1 cells wereco-transfected with the vectors for MT1-MMP and MT1PEX-F at theindicated DNA ratio, and reacted with the purified proMMP-2. The culturesupernatant was analyzed for MMP-2 by gelatin zymography (upper panel),and the cell lysates were analyzed by Western blotting using ananti-MT1-PEX antibody (mid panel, Whole cells). The transfected cellswere subjected to surface biotinylation, and analyzed by Westernblotting using an anti-MT1-PEX (lower panel, Surface Biotinylation).

[0083]FIG. 29: As in the case with FIG. 28, the binding of proMMP-2 tothe transfected cells was examined. The samples were analyzed by gelatinzymography.

[0084]FIG. 30: As in the case with FIG. 28, the transfected cells werecultured on each slide glass coated with Alexa488-labeled gelatin andthen examined for their in situ gelatin degrading activity. The gelatindegrading activity was visualized as the dark non-fluorescent area. Bar:50 μm.

[0085]FIG. 31: In regard to the efficacy of MT1PEX-F on the matrigelinvasive action of HT1080 cells, HT1080 cells were transfected with thestable expression plasmids for MT1PEX-F and the empty vector (Mock).Hygromycin resistant cells were collected and re-seeded. The culturemedium was changed to a serum-free medium in the presence or absence ofBB94, and the cells were further cultured. The culture media and thecell lysates were analyzed by gelatin zymography and Western blottingusing an anti-MT1PEX antibody, respectively.

[0086]FIG. 32: As in the case with FIG. 31, HT1080 cells weretransfected with the expression plasmids for MT1PEX-F or the emptyvector. Endogenous MT1-MMP and MT1PEX-F in the transfected cells werestained immunologically using a rabbit anti-MT1Cat antibody and ananti-FLAG M1 antibody. A white arrow indicates a site where signals forendogenous MT1-MMP (Anti-MT1Cat) and MT1PEX-F (Anti-FLAG M1) wereco-localized. Bar: 50 μm.

BEST MODES FOR CARRYING OUT THE INVENTION

[0087] According to the present invention, inhibitory methods for theactivation of proMMP-2 relying on inhibiting the formation of a complexcomprised of plural MT1-MMPs, for example, the formation of a MT1-MMPhomodimer wherein said MT1-MMP is present on the cell surface andsubstances each having activity therefor can be provided. Further,screening methods for such active substances and pharmaceutical productsuseful in such applications are provided.

[0088] Particularly, the PEX domain of MT1-MMP plays an important rolein the homodimer formation of MT1-MMP. For instance, PEX lacking thecatalytic domain of MT1-MMP shows an in vivo and in vitro inhibitoryactivity on the homodimer formation of MT1-MMP. Therefore, it ispromising as an inhibitor or pharmaceutical drug, and enablesdevelopment of various inhibitors against the homodimer formation ofMT1-MMP based on this fact. In another aspect, the present inventionprovides techniques for monitoring the homodimer formation of MT1-MMP.Furthermore, it also provides an outlook for gene therapy.

[0089] The present invention relates to antibodies, for example,monoclonal antibodies which specifically recognize the PEX domain ofMT1-MMP, and provides immunological assay reagents, pharmaceuticals andfurther various assay techniques using said antibody.

[0090] The term “PEX” or “PEX protein” used herein may cover MT1-MMP PEXdomain proteins or fragments thereof [for example, from amino acidresidues 316 to 508, selected from the amino acid sequence of MT1-MMP(SWISS-PROT accession number: P50281), and in some cases, from aminoacid residues 319 to 535 of said MT1-MMP]. They may also be mutants,analogs and derivatives derived or constructed as described hereinbelow. It is not particularly limited as long as it possesses a bindingactivity to the PEX domain of MT1-MMP. Such substances include thosehaving an inhibitory activity on the formation of complexes comprised ofmultiple MT1-MMPs, and preferably those having an inhibitory activity onthe homodimer formation of MT1-MMP present on the cell surface. Therepresentatives include, but are not limited to, those which lack thesequence from Ser² to Ile³¹⁸, selected from the amino acid sequence fromMet¹ to Gly⁵³⁵ of MT1-MMP, i.e., MT1PEX. The PEX protein may includecorresponding PEX proteins with mutations (such as mutated PEX proteinswherein one or more amino acid residues are appropriately substituted,deleted, inserted, rearranged or added) introduced into the naturallyoccurring amino acid sequence of proteins each having an amino acidsequence corresponding to the PEX domain of MT1-MMP as long as theproteins have a desired biological activity (for instance, an inhibitoryactivity against the homodimer formation of MT1-MMP or an inhibitoryactivity against the proMMP-2 activation via inhibition of MT1-MMPhomodimer formation, or further a suppressing or inhibiting activity oncell migration, invasion and/or metastasis resulting from the homodimerformation of MT1-MMP, etc.).

[0091] Such mutations, conversions and modifications include chemicaltechniques or those applied with the standard gene engineering methodsbased on the nucleotide sequence of the MT1-MMP gene (GenBank™/EMBLaccession number: D26512). For example, they are those described in“Zoku-Seikagaku Jikken Koza 1, Idenshi Kenkyuhou II” JapaneseBiochemical Society ed., p. 105 (Susumu Hirose), Tokyo Kagaku-dojinPublishing Co., Inc. (1986); “Shin-Seikagaku Jikken Koza 2, Kakusan III(Recombinant DNA technique)” Japanese Biochemical Society ed., p. 233(Susumu Hirose), Tokyo Kagaku-dojin Publishing Co., Inc. (1992);“Methods in Enzymology” Vol. 154, pp. 350 & 367, R. Wu, L. Grossman ed.,Academic Press, New York (1987); “Methods in Enzymology” Vol. 100, pp.457 & 468, R. Wu, L. Grossman ed., Academic Press, New York (1983); J.A. Wells, et al., Gene, 34:315, 1985; T. Grundstroem et al., NucleicAcids Res., 13: 3305, 1985; J. Taylor et al., Nucleic Acids Res., 13:8765, 1985; “Methods in Enzymology” Vol. 155, p. 568, R. Wu ed.,Academic Press, New York (1987); A. R. Oliphant et al., Gene, 44:177,1986 etc. For example, included are methods such as the site-directedmutagenesis method (site specific mutagenesis method) utilizingsynthetic oligonucleotides (Zoller et al., Nucl. Acids Res., 10:6487,1987; Carter et al., Nucl. Acids Res., 13:4331, 1986), the cassettemutagenesis method (Wells et al., Gene, 34:315, 1985), restrictionselection mutagenesis method (Wells et al., Philos. Trans. R. Soc.London Ser A, 317: 415, 1986), the alanine scanning method (Cunningham &Wells, Science, 244: 1081-1085, 1989), PCR mutagenesis method, Kunkelmethod, dNTP[α S] method (Eckstein), the region directed mutagenesismethod using sulfurous acid and nitrous acid and the like.

[0092] Moreover, in the proteins obtained, amino acid residues containedtherein can be modified by chemical techniques, and for example, can bemodified and partially decomposed to make derivatives thereof usingenzymes such as peptidases, e.g., pepsin, chymotrypsin, papain,bromelain, endopeptidase, exopeptidase and the like. Also, the proteincan be glycosylated or deglycosylated, or glycosylation sites can bealtered in vivo or in vitro (WO 87/05330; Apin and Wriston, CRC Crit.Rev. Biochem., 259-306, 1981; Hakimuddin et al., Arch. Biochem.Biophys., 259:52, 1987; Edge et al., Anal. Biochem., 118:131, 1981;“Methods in Enzymology” Vol. 138, p. 350, Academic Press, New York,1987, etc.).

[0093] In the proteins of the present invention, the C-terminal end istypically a carboxyl group (—COOH) or a carboxylate (—COO⁻), but theC-terminal end may be an amide form (—CONH₂) or an ester form (—COOR).For said ester, R includes C₁ to C₆ alkyl groups such as methyl, ethyl,n-propyl, isopropyl and n-butyl, C₃ to C₈ cycloalkyl groups such ascyclopentyl and cyclohexyl, C₆ to C₁₂ aryl groups such as phenyl andα-naphthyl, phenyl-C₁ to C₂ alkyl groups such as benzyl and phenethyl,C, to C₁₋₄ aralkyl groups including α-naphthyl-C₁ to C₂ alkyl groupssuch as α-naphthylmethyl, as well as a pivaloyloxymethyl group widelyused as an ester in oral use. When the proteins of the present inventionhave a carboxyl group (or carboxylate) at the site other than theC-terminal end, those in which the carboxyl group can be amidated oresterified are included in the proteins of the present invention. As theester in this case, for example, the C-terminal ester and the likedescribed above are used.

[0094] Additionally, the proteins of the present invention may be thosehaving a methionine residue at N-terminus in the above proteins, andfurther include those in which an amino group of the methionine residueis protected with a protecting group (for example, C₁ to C₆ acyl groupsincluding C₁ to C₅ alkyl-carbonyl groups such as formyl and acetyl),those in which the N-terminus is cleaved in vivo and the resultantglutamyl group is pyroglutamylated, those in which substituents (forexample, —OH, —COOH, amino, imidazole, indole, guanidino groups and thelike) on side chains of the intramolecular amino acids are protectedwith appropriate protecting groups (for example, C₁ to C₆ acyl groupssuch as formyl and acetyl groups), or conjugated proteins (such asso-called glycoproteins) in which saccharide chains are linked. And, theproteins may be expressed as fusion proteins when made by generecombination methods, and may convert or process into those havingsubstantially equivalent biological activity as compared to those whichnaturally occur in vivo or in vitro. The production methods by fusionusually used in gene engineering can be used. Such fusion proteins canbe purified by an affinity chromatography taking advantage of theirfusion moieties.

[0095] Such fusion proteins include those fused to a histidine tag, orthose fused to the amino acid sequence of maltose-binding protein (MBP),glutathione S-transferase (GST) or thioredoxin (TRX). Similarly, thepolypeptide can be added with a tag of heterogenous epitope, and can beisolated/purified by an immunoaffinity chromatography using an antibodyspecifically binding to the epitope. The representatives include polyhistidine (poly-His) or a polyhistidine-glycine (poly-His-Gly) tag, fluHA tag and the antibody thereto, 12CA5, c-Myc tag and the antibodiesthereto, 8F9, 3C7, 6E10, G4, B7 and 9E10, Herpes Simplex virusglycoprotein D (gD) tag and the antibody thereto, FLAG peptide, KT3epitope peptide, α-tubulin epitope peptide, T7 gene 10 protein peptidetags and the like (Field et al., Molecular and Cellular Biology 8:2159-2165, 1988; Evans et al., Molecular and Cellular Biology 5:3610-3616, 1985; Paborsky at al., Protein Engineering 3(6): 547-553,1990; Hopp et al., BioTechnology 6: 1204-1210, 1988; Martin et al.,Science 255: 192-194, 1992; Skinner et al., J. Biol. Chem., 266:15163-15166, 1991; Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87: 6393-6397, 1990). The expression and purification of such fusionproteins can be performed using commercially available kits suitable forthese techniques, and can also be carried out according to protocols asinstructed by manufacturers or distributors of the kits.

[0096] Modifications and alterations of the protein structures can beperformed in reference to “Shin-Seikagaku Jikken Koza 1, Protein VII,Protein Engineering” Japanese Biochemical Society ed., (TokyoKagaku-dojin Publishing Co., Inc., 1993) using the methods describedtherein or the methods described in the references cited therein, and,further, the methods substantially equivalent thereto. Their biologicalactivities as described herein below may include an immunologicalactivity, for example, an antigenicity. The modification and alterationmay be deamination, hydroxylation, phosphorylation, methylation,acetylation, ring-opening, cyclization, alteration of containedsaccharide chains to different types, increasing or decreasing thenumber of contained saccharide chains, substitution to D-amino acidresidues, etc. Those methods are known in the art (for example, T. E.Creighton, Proteins: Structure and Molecular Properties, pp. 79-86, W.H. Freeman & Co, San Francisco, USA, 1983, etc.).

[0097] Thus, the proteins may be those of which one or more amino acidresidues are different from those which naturally occur in terms ofidentity or those of which sites of one or more amino acid residues aredifferent from those which naturally occur. The proteins includedeletion analogs where one or more (for example, from 1 to 190,preferably from 1 to 50, more preferably from 1 to 20, still preferablyfrom 1 to 10 and especially from 1 to 5) amino acid residuescharacteristic of PEX are deleted, substituted analogs where one or more(for example, from 1 to 100, preferably from 1 to 50, more preferablyfrom 1 to 20, still preferably from 1 to 10 and especially from 1 to 5)amino acid residues characteristic of PEX are substituted with otherresidues, and additional analogs where one or more (for example, from 1to 100, preferably from 1 to 50, more preferably from 1 to 20, stillpreferably from 1 to 10 and especially from 1 to 5) amino acid residuesare added. The mutants as the above are all included in the presentinvention as long as the domain structure or active center structurecharacteristic of native PEX is retained and the resistance is retainedagainst the dimer formation of MT1-MMP present on a cell surfacedescribed above. Also, it is thought that the proteins may include thosehaving parts of primary structure conformation substantially equivalentto that of native PEX. Furthermore, it is also thought that the proteinsmay include those having biological activity substantially equivalent tonative PEX except for being resistant to the dimer formation of MT1-MMPpresent on a cell surface.

[0098] Moreover, the protein can be one of the mutants which naturallyoccur. The proteins include those having more than 50% homology, andmore preferably 70% or more or 90% or more of an homologous amino acidsequence for the amino acid sequence ranging from the 316th amino acidresidue P to the 508th amino acid residue C (and/or the amino acidsequence ranging from the 319th to 535th amino acid residues) in theamino acid sequence of MT1-MMP. The fragments derived from the proteinare parts of peptides of the PEX protein (i.e., partial peptides of thePEX protein), and may be any of those as long as they have activitysubstantially equivalent to the above PEX protein. For example, thepartial peptides of the protein of the present invention includepeptides having at least two or more consecutive, preferably 5 or more,more preferably 10 or more, still preferably 20 or more, still morepreferably 50 or more, and, in some cases, 100 or more amino acidsequences in the constitutive amino acid sequence ranging from the 316thto the 508th amino acid residues (and/or the 319th to the 535th aminoacid residues) of the amino acid sequence of MT1-MMP. For example,included are those having the same homology as described above in regardto the homology to the region corresponding to the amino acid sequenceof MT1PEX.

[0099] The term “substantially equivalent” used herein means that theprotein activity, for example, the resistance to proMMP-2 activation bythe homodimer formation of MT1-MMP, the physiological activity orbiological activity thereto is substantially equivalent. Furthermore,the meanings of that term may include a case having substantiallyhomogenous activity. The substantially homogenous activity can include,for example, a predisposition to inhibit the dimer formation, and theactivity to suppress and/or inhibit cell migration, invasion and/ormetastasis associated with the activation of proMMP-2. The substantiallyhomogenous activity indicates that these activities are qualitativelyhomogenous, and, for example, that they are physiologically,pharmacologically or biologically homogenous. For instance, it ispreferred that the activities to inhibit the dimer formation areequivalent (for example, from about 0.001 to 1000 fold, preferably fromabout 0.01 to 100 fold, more preferably from about 0.1 to 20 fold, andstill preferably from about 0.5 to 2 fold), but quantitative elementssuch as the extents of these activities, molecular weights of theproteins etc. may be different.

[0100] Next, the substitution, deletion or insertion of amino acids doesnot often cause a great alteration in physiological or chemicalproperties of a polypeptide. In such a case, a polypeptide withsubstitution, deletion or insertion will be considered to besubstantially identical to a polypeptide without such substitution,deletion or insertion. Substantially identical substituents of aminoacids in the amino acid sequence can be selected from other amino acidsin the class to which the amino acid belongs. For instance, non-polar(hydrophobic) amino acids include alanine, phenylalanine, leucine,isoleucine, valine, proline, tryptophan, methionine and the like, polar(neutral) amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine, glutamine and the like, amino acids havingpositive charge (basic amino acids) include arginine, lysine, histidineand the like, and amino acids having a negative charge (acidic aminoacids) include aspartic acid, glutamic acid and the like.

[0101] The methods known in the peptide synthetic art, for example,chemical synthetic methods such as liquid phase synthetic methods, andsolid phase synthetic methods can be used for the synthesis of theproteins and partial peptides thereof of the present invention (Stewartet al., Solid-Phase Peptide Synthesis, W. H. Freeman Co., San Francisco,Calif., USA, 1969; Merrifield, J. Am. Chem. Soc., 85:2149-2154, 1963; J.Org. Chem., 37:3404, 1972; G. B. Fields (ed), “Methods in Enzymology”,Vol. 289 (Solid-Phase Peptide Synthesis), Academic Press, New York,1997). In such methods, using a resin for synthesis of proteins orpeptides, an appropriately protected amino acid is sequentially attachedto the desired amino acid sequence on the resin by various condensationmethods as known in the art. Various activation reagents as known in theart are preferably used for the condensation reactions. For example,carbodiimides such as dicyclohexylcarbodiimide can be preferably used assuch reagents. An objective reagent can be obtained by appropriatelyeliminating a protecting group when a product has the protecting group.

[0102] The proteins and partial peptides thereof of the presentinvention can be converted to salts thereof according to the methodsknown in the art or methods based thereon when they are obtained as freetypes. Also, when they are obtained as salt types, they can be convertedto free types or other salt types according to the methods known in theart or methods based thereon.

[0103] The proteins of the present invention (including the proteinfragments having the given consecutive regions of the amino acidsequence present in MT1-MMP and the fragments derived therefrom, theproteins and the fragments derived therefrom which are obtained fromthose having a deletion of one selected from the group consisting of asignal peptide domain, propeptide domain, core enzymatic domain, hingedomain, and transmembrane domain of MT1-MMP) can be obtained by applyinggene engineering techniques based on the base sequence of human MT1-MMPgene (GenBank™/EMBL accession number: D26512). Nucleic acids having thebase sequence of the human MT1-MMP gene, for example the DNA moleculecan be obtained by PCR or ligase chain reaction (LCR) amplification ofthe desired sequence by designing and synthesizing appropriate primersbased on the MT1-MMP base sequence, preferably using mRNA or cDNAprepared from the mRNA derived from the animal which is an origin of thehuman MT1-MMP base sequence. In some cases, the DNA molecule can beobtained by screening human genomic DNA libraries or cDNA librariesderived from humans constructed from various human tissues or culturedcells using the resultant DNA fragments as probes.

[0104] First, sense and antisense primers are synthesized based on thebase sequence of the MT1-MMP. The primers used for the PCR method arenot particularly limited as long as they can amplify the DNA fragmentscontaining the desired site. The sense primer can be synthesizedpreferably by selecting the 5′-terminal exon site of the gene, and theantisense primer can be synthesized preferably by selecting the3′-terminal exon site of the gene, and more preferably can be selectedfrom the sites other than the exon site utilized for synthesis of thesense primer. It is preferred that the 5′-terminal primer is selected tocontain at least an initiation codon or to be able to amplify includingthe initiation codon and that the 3′-terminal primer is selected tocontain at least a stop codon or to be able to amplify including thestop codon. Representatively, the primers can use (a) oligonucleotideshaving base sequences corresponding to the given 5′-terminal site of thebase sequence of human MT1-MMP: GenBank™/EMBL accession number: D26512,and (b) oligonucleotides having complementary base sequences for thegiven 3′-terminal site of the base sequence of the human MT1-MMP.

[0105] It may be targeted to obtain cDNA for the gene as its full lengthat a time. However, plural primers can be designed and synthesized withutilization of the given exon sites (plural exon sites) and plural PCRsmay be designed and conducted, whereby DNA fragments may be obtained.The target cDNA of said gene can be obtained from DNA fragments clonedon the basis of said resultant DNA fragments. The primers includeoligonucleotides preferably composed of 5 or more nucleotide bases, forexample, oligonucleotides composed of 10 to 50, more preferably 15 to35, and still preferably 18 to 25 nucleotide bases. Synthesis of theprimers can be carried out by methods known in the art, and, forexample, the primers can be synthesized by the phosphodiester, thephosphotriester, and the phosphoamidite methods using, for example, anautomatic DNA synthesizer, e.g., model 381A DNA synthesizer, AppliedBiosystems.

[0106] mRNA samples can be isolated from various human tissues andcultured cells known to express human MT1-MMP (in particular, humancells obtained from a human heart, brain, placenta, lung, liver,skeletal muscle, kidney, pancreas and the like, tumor cell lines such ashuman lung epidermoid cancer cell lines and the like). Isolation of mRNAcan be carried out by the methods known in the art or methodssubstantially equivalent thereto or modified methods thereof, but canalso be performed by methods described in J. Sambrook et al., “MolecularCloning”, 2nd ed., Chapter 7, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989; D. M. Glover et al., ed., “DNA Cloning”, 2nd ed.,Vol. 1, (The Practical Approach Series), IRL Press, Oxford UniversityPress, 1995; L. Grossman et al. ed., “Methods in Enzymology”, Vol. 12,Part A & B, Academic Press, New York, 1968; S. L. Berger et al. ed.,“Methods in Enzymology”, Vol. 152, p.33 & p.215, Academic Press, NewYork, 1987; Biochemistry, 18:5294-5299, 1979 and the like, for example,the guanidine cesium chloride method, the thiocyanate guanidine method,the phenol method and the like. Kits used for the isolation of mRNAinclude those commercially available from Pharmacia, Stratagene,Gibco-BRL etc. Poly (A)⁺ mRNA can be obtained by purifying the obtainedtotal RNA using an oligo (dT) cellulose column, spin column, oligo (dT)binding magnetic beads and the like, if necessary.

[0107] The cDNA is made using this mRNA and reverse transcriptase (RNAdependent DNA polymerase). An oligo (dT) primer can also be used in thereverse transcription reaction. Those preferably having 12 to 18 Tresidue can be used as the oligo (dT) primers. It is also preferred thata synthetic oligonucleotide primer where a restriction enzyme site isligated to the 5′ side of the 12 to 18 T residue when directionalcloning is carried out. Examples of such primers include Xba I oligo(dT) primer adapter and the like. When random hexamer primers are used,a probability to obtain the 5′-terminal site of mRNA is increased, andthe random hexamer primer can be used alone or in combination with theoligo (dT) primer. In particular, it is also preferred thatoligonucleotide primers synthesized based on the base sequence of theabove human MT1-MMP gene (GenBank™/EMBL accession number: D26512) areused. A RNA inhibitor can also be added in the reverse transcriptionreaction if necessary. The synthesis of cDNA using mRNA and the reversetranscriptase can be carried out by methods known in the art or methodssubstantially equivalent thereto and modified methods thereof, whichinclude methods described in H. Land et al., Nucleic Acids Res., 9:2251,1981; U. Gubler et al., Gene, 25:263-269, 1983; S. L. Berger et al. ed.,“Methods in Enzymology”, Vol. 152, p.307, Academic Press, New York,1987.

[0108] Polymerase chain reaction (PCR) is carried out using theresultant cDNA as well as the aforementioned sense and antisense primersto amplify the desired region of the cDNA.

[0109] The term “polymerase chain reaction” or “PCR” used herein usuallyrefers to techniques described in U.S. Pat. No. 4,683,195. For example,the PCR is an in vitro method for the enzymatic amplification of desiredspecific nucleotide sequences. In general, the PCR includes repetitiveseries of cycles wherein a primer elongation synthesis is constructedusing two oligonucleotide primers capable of preferentially hybridizingwith a template nucleic acid. Typically, the primers used in PCR mayinclude those which are complementary to the internal nucleotidesequence of interest in the the template. For example, preferable primerpairs as used herein may be those which are complementary to both endsof said nucleotide sequence to be amplified, or flanking regionsadjacent to said nucleotide sequence.

[0110] A template (for example, DNA synthesized using, as a template,mRNA) and primers synthesized according to designs on said gene aremixed with a 10× reaction buffer (attached with a Taq DNA polymerasekit), dNTPs (deoxyribonucleoside triphosphates; dATP, dGTP, dCTP anddTTP mix), Taq DNA polymerase and deionized distilled water. The mixtureis subjected to 25 to 60 cycles of amplification using an automatedthermal cycler such as GeneAmp 2400 PCR system, Perkin-Elmer/Cetus undergeneral PCR cycle conditions. The number of amplification cycles can besuitably set to an appropriate value depending on purposes. The PCRcycle includes, for example, denaturation at 90 to 95° C. for 5 to 100sec, annealing at 40 to 60° C. for 5 to 150 sec and extension at 65 to75° C. for 30 to 300 sec, and preferably denaturation at 94° C. for 15sec, annealing at 58° C. for 15 sec and extension at 72° C. for 45 sec.For the annealing temperature and reaction time, an appropriate value issuitably selected by experimentation. For the denaturation and extensiontime, an appropriate value suitably varies according to the strandlength of expected PCR products. In general, the time of annealingpreferably varies depending on the Tm value of primer-template DNAhybrids. The time period of extension usually is set with the aim ofgetting about 1 min per 1000 bp in strand length, but it may be possibleto select a shorter time period in some cases.

[0111] The resultant PCR products are typically subjected toelectrophoresis on 1 to 2% agarose gels. Specific bands are cut out fromthe gel, and DNA is extracted with a commercially available kit, e.g.,Gene clean kit (Bio 101) and the like. The extracted DNA is cleaved withappropriate restriction enzymes and purified if necessary. Then, the5′-end is, if necessary, phosphorylated with T4 polynucleotide kinase,etc. and subsequently the DNA is ligated into an appropriate plasmidvector including a pUC vector such as pUC18, and transformed intosuitable competent cells. The cDNA library can also be constructed basedon the produced DNA fragments using phage vectors, plasmid vectors etc.Transformation of host cells such as E. coli can be carried out bymethods known in the art such as the calcium method, therubidium/calcium method, the calcium/manganese method, the TFB highefficiency method, the FSB frozen competent cell method, the rapidcolony method, the electroporation and the like or methods substantiallyequivalent thereto (D. Hanahan, J. Mol. Biol., 166:557, 1983, etc.).

[0112] Nucleic acids having an entire or partial base sequence of thegene can also be obtained by chemical synthesis. In such cases, thesemay be made by chemically synthesizing the fragments and ligating themwith enzymes. Also, the desired sequence can be obtained using thechemically synthesized fragments as primers or probes.

[0113] The PCR reactions can be carried out by methods known in the artor methods substantially equivalent thereto and modified methodsthereof, and can be performed according to methods described, forexample in Saiki, R., et al., Science, 230:1350, 1985; Saiki, R., etal., Science, 239:487, 1988; Erlich, H. A. ed., PCR Technology, StocktonPress, 1989; Glover, D. M., et al. ed., “DNA Cloning”, 2nd ed., Vol. 1,(The Practical Approach Series), IRL Press, Oxford University Press,1995; Innis, M. A. et al. ed., “PCR Protocols: Guide to Methods andApplications”, Academic Press, New York, 1990; McPherson, M. J., Quirke,P. and Taylor, G. R. ed., PCR: Practical Approach, IRL Press, Oxford,1991; Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA, 85:8998-9002,1988, and modified or altered methods thereof. The PCR methods can alsobe performed using commercially available kits suitable therefor, andcan also be carried out according to protocols described bymanufacturers or distributors of the kits.

[0114] The term “oligonucleotide(s)” used herein refers to a relativelyshort single-stranded polynucleotide or double-stranded polynucleotides,or preferably polydeoxynucleotide(s), They can be chemically synthesizedby known methods as described in Angew, Chem. Int. Ed. Engl., 28:716-734, 1989, including the triester method, the phosphite method, thephosphoamidite method, the phosphonate method and the like. It has beentypically known that the synthesis can be conveniently carried out onmodified solid supports. For example, the synthesis can be carried outusing an automated synthesizer and such a synthesizer is commerciallyavailable. The oligonucleotide may contain one or more modifiednucleotide bases and, for example, it may contain a nucleotide basewhich does not naturally occur, such as inosine, or a tritylatednucleotide base.

[0115] The DNA fragments encoding the desired human MT1-MMP can beyielded by cloning the resultant PCR product and identifying theresultant PCR product. Also, the target DNA can be isolated by screeningvarious cDNA libraries using the DNA fragment as a probe. Commerciallyavailable plasmid vectors such as p-Direct (Clontech), pCR-Script™ SK(+)(Stratagene), pGEM-T (Promega), pAmp™ (Gibco-BRL) etc., can be used forcloning of the PCR products.

[0116] In order to yield the full length gene sequence containing thefull open reading frame, the cDNA libraries constructed from varioushuman tissues or cultured cells as mentioned above are screened usingthe DNA fragments obtained as described above as probes, cloneshybridizing to the probes are selected, an insertion base sequence ofcDNA in the clone is sequenced, and the DNA fragment composing the geneis identified if necessary. Of course, the insertion sequence of cDNA inthe clone can be subcloned if necessary. To label the probe and the likewith radioisotopes and the like, it can be carried out usingcommercially available kits such as a random primed DNA labeling kit(Boehringer Mannheim) and the like.

[0117] Reverse transcription PCR (polymerase chain reaction coupledreverse transcription; RT-PCR) and RACE (rapid amplification of cDNAends) can be applied to isolate the target DNA. RACE can be carried outaccording to, for example, the methods described in Innis, M. A., etal., ed., “PCR Protocols” (Frohman, M. A., “A Guide to Methods andApplications”), pp.28-38, Academic Press, New York, 1990 etc. RT-PCRproducts can be cloned into plasmid vectors, which can be introducedinto highly efficient competent cells.

[0118] Furthermore, cDNA can also be PCR-amplified by utilizing themethod where mRNA can be isolated and purified from a trace quantity oftissue or cells, for example, the method by utilizing commerciallyavailable kits such as the REX kit (United States Biochemical), GlassMAX™ RNA spin cartridge system (Gibco-BRL) and the like, reverselytranscripting the resultant mRNA using an oligo (dT) primer tosynthesize the first strand DNA, then attaching a homopolymer tail (forexample, G residue) to the 3′-terminus of the first strand DNA orattaching an adapter to the DNA, and subsequently using the oligo (dT)primer and an oligo (dC) primer or the adapter primer. The commerciallyavailable kits suitable for this include SuperScript™ pre-amplificationsystem (Gibco-BRL), cDNA Cycle™ kit (Invitrogen) and the like.

[0119] Hybridization is carried out by transferring a sample (forexample, gene libraries prepared from human cells and tissues,representatively, those obtained by constructing into a phage such as λgt10 and the like, which is infected to host E. coli such as the E. coliC600hfl strain to form plaques) on a membrane such as a nylon filter,performing denaturation, fixation and washing treatments if necessary,and subsequently reacting the sample transferred on the membrane with alabeled probe DNA fragment denatured, if necessary, in the buffer forhybridization.

[0120] The hybridization treatment is carried out usually at about 35°C. to about 80° C., more suitably from about 50° C. to about 65° C. forabout 15 min to about 36 hrs, more suitably from about 1 hr to about 24hrs, but can be carried out by selecting an optimal conditionappropriately. For instance, the hybridization treatment is carried outat about 55° C. for about 18 hrs. As buffers for the hybridization,those that can be used are selected among those usually used in the art,for example, Rapid hybridization buffer (Amersham) and the like can beused. The denaturation treatment of the transferred membrane includesthe method using an alkali denaturation solution, and it is preferredthat treatment is carried out with a neutralization solution or bufferafter the treatment. The fixation treatment of the membrane is carriedout by baking usually at about 40° C. to about 100° C., more suitablyfrom about 70° C. to about 90° C. for about 15 min to about 24 hrs, moresuitably from about 1 hr to about 4 hrs, but can be carried out byselecting an optimal condition appropriately. For instance, the fixationis carried out by baking the filter at about 80° C. for about 2 hrs. Thewashing treatment of the transferred membrane can be carried out bywashing with a washing solution which is usually used in the art, forexample 50 mM Tris-HCl buffer, pH 8.0 containing 1M NaCl, 1 mM EDTA and0.1% sodium dodecyl sulfate (SDS), and the like. As for membranes suchas a nylon filter, those that can be used are those usually selectedfrom the art, and can include, for example, the nylon filter [Hybond-N,Amersham] and the like.

[0121] The above alkali denaturation solution, neutralization solutionand buffer can be used by selecting those usually used in the art. Thealkali denaturation solution can include, for example, the solutioncontaining 0.5 M NaOH and 1.5 M NaCl and the like, the neutralizationsolution can include, for example, 0.5 M Tris-HCl buffer, pH 8.0containing 1.5 M NaCl and the like, and the buffer can include, forexample, 2×SSPE (0.36 M NaCl, 20 mM NaH₂PO₄ and 2 mM EDTA) and the like.

[0122] It is preferred that prehybridization treatment is performed forthe transferred membrane prior to the hybridization treatment, ifnecessary, to prevent non-specific hybridization reactions. Theprehybridization treatment can be carried out, for example, by immersingin the prehybridization solution [50% formamide, 5× Denhardt's solution(0.2% bovine serum albumin, 0.2% polyvinyl pyrrolidone), 5×SSPE, 0.1%SDS, 100 μg/ml heat denatured salmon sperm DNA] and reacting from about35° C. to about 50° C., preferably at about 42° C. for about 4 to 24hrs, preferably from about 6 to 8 hrs, but those skilled in the art candetermine more preferable conditions by appropriately repeatingexperiments for these conditions. The denaturation of the labeled probeDNA fragments used for the hybridization can be carried out by, forexample, heating at about 70° C. to 100° C., preferably at 100° C. forabout 1 min to about 60 min, preferably for about 5 min. Thehybridization can be carried out by methods known in the art or methodsfollowing thereto. Herein, a stringent condition indicates, for example,the condition where the concentration of sodium is about 15 to 50 mM,preferably about 19 to 40 mM, more preferably about 19 to 20 mM, and thetemperature is about 35 to 85° C., preferably about 50 to 70° C., morepreferably about 60 to 65° C.

[0123] After completion of the hybridization, the filter is thoroughlywashed to remove labeled probes other than the labeled probe DNAfragments having the specific hybridization reaction. The washing of thefilter can be carried out by selecting among those usually used in theart and, for example, can be performed by washing with a 0.5×SSCsolution (15 mM citrate buffer, pH 7.0 containing 0.15 M NaCl)containing 0.1% SDS.

[0124] Hybridized plaques can be detected representatively byautoradiography, but those appropriately selected among the methods usedin the art can also be used for detection of plaques. The targetrecombinant phages are obtained from cultured E. coli by suspending theplaques corresponding to the detected signals in the appropriate buffer,e.g., SM solution (50 mM Tris-HCl buffer, pH7.5 containing 100 mM NaCland 10 mM MgSO₄), diluting the phage suspension moderately to infect theE. coli, and culturing the obtained E. coli. The treatment can berepeatedly carried out if necessary in which the target recombinantphages are screened in genes or cDNA libraries by the hybridizationtreatment using the above probe DNA. The target recombinant phages canbe yielded from the cultured E. coli by conducting an extraction,centrifugation treatments etc.

[0125] The yielded phage particles can be isolated and purified bymethods usually used in the art and, for example, can be purified by theglycerol gradient ultracentrifuge method (Maniatis, T. ed., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2nd ed.,78, 1989) etc. The DNA can be isolated and purified from the phageparticles by methods usually used in the art and, for example, isobtained by suspending the yielded phages in TM solution (50 mM Tris-HClbuffer, pH 7.8 containing 10 mM MgSO₄), treating with DNase I and RNaseA and the like, then adding a mixture solution of 20 mM EDTA, 50 μg/mlproteinase K and 0.5% SDS, incubating at about 65° C. for about onehour, extracting with phenol and diethylether, precipitating DNA byethanol precipitation, subsequently washing the yielded DNA with 70%ethanol, and allowing to dry and dissolving in TE solution (10 mMTris-HCl buffer, pH 8.0 containing 10 mM EDTA). Also, the target DNA canbe obtained in a large amount by subcloning and, for example, thesubcloning can be carried out by using E. coli as the host using plasmidvectors and the like. The obtained DNA by such a subcloning can also beisolated and purified similarly as in the above by methods such ascentrifugation, phenol extraction, ethanol precipitation etc.

[0126] Chimera DNA and the like containing human MT1-MMP DNA, TIMP-2DNA, MMP-2 DNA, nerve growth factor receptor (NGFR) DNA and partsthereof are obtained in the above ways. Others such as nucleic acids forDNA encoding the desired polypeptides can be obtained if necessary.According to the present invention, obtained are the DNA such as the DNAof human MT1-MMP with the addition of a FLAG epitope with deletion of acatalytic region, the DNA of human MT1-MMP with the addition of the FLAGepitope with deletion of PEX, chimera mutants of human MT1-MMP DNA andNGFR DNA with the addition of the FLAG epitope, chimera mutants of humanMT1-MMP DNA and NGFR DNA with the addition of the FLAG epitope withdeletion of the catalytic region, chimera mutants of human MT1-MMP DNAand NGFR DNA with the addition of the FLAG epitope with deletion of PEX,the DNA encoding from Cys³³⁶ to Gly⁵⁵⁰ of Met plus MT1-MMP and the like.The representative chimera mutants include those in which TM/CP of humanMT1-MMP is substituted with TM/CP of NGFR.

[0127] The nucleic acids obtained in the present invention are singlechain DNA, double chain DNA, RNA, DNA:RNA hybrids, synthetic DNA and thelike, and may be either of human genomic DNA, human genomic DNAlibraries, cDNA derived from human tissues and cells and synthetic DNA.The nucleic acids may be those which hybridize to those having thecharacteristic sequence specifically disclosed herein in the stringentcondition. The base sequence of the nucleic acids is modified (e.g.,addition, elimination, substitution etc.) by the methods known by thoseskilled in the art such as the gene recombination techniques asmentioned above such that it encodes the desired protein.

[0128] The gene recombination techniques can be carried out by themethods described in, for example, Sambrook, J., Fritsch, E. F. &Maniatis, T., “Molecular Cloning: A Laboratory Manual (2nd ed.)”, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Glover,D. M. et al. ed., “DNA Cloning”, 2nd ed., Vol. 1 to 4 (The PracticalApproach Series), IRL Press, Oxford University Press, 1995;“zoku-Seikagaku Jikken Koza 1, Idenshi Kenkyuhou II” ed., by JapaneseBiochemical Society, Tokyo Kagaku-dojin Publishing Co., Inc., 1986;“Shin-Seikagaku Jikken Koza 2, Kakusan III (Recombinant DNA technique)”ed., by Japanese Biochemical Society, Tokyo Kagaku-dojin Publishing Co.,Inc., 1992; Wu, R., ed., “Methods in Enzymology”, Vol. 68 (RecombinantDNA), Academic Press, New York, 1980; Wu, R. et al., ed., “Methods inEnzymology”, Vol. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA,Part C), Academic Press, New York, 1983; Wu, R. et al., ed., “Methods inEnzymology”, Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA,Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York, 1987;Miller, J. H., ed., “Methods in Enzymology”, Vol. 204, Academic Press,New York, 1991; Wu, R. et al., ed., “Methods in Enzymology”, Vol. 218,Academic Press, New York, 1993; Weissman, S., ed., “Methods inEnzymology”, Vol. 303, Academic Press, New York, 1999; Clorioso, J. C.,ed., “Methods in Enzymology”, Vol. 306, Academic Press, New York, 1999etc., or by methods described in the references cited therein or methodssubstantially equivalent thereto or modified methods thereof (thedescriptions therein are incorporated in the disclosure of the presentinvention as reference).

[0129] The determination of the base sequence can be carried out usingthe dideoxy methods such as the M13 dideoxy method, Maxam-Gilbert methodand the like and, for example, can be carried out using commerciallyavailable sequencing kits such as the Taq dye primer cycle sequencingkit, Sequenase v 2.0 kit and the like, or using an automatic sequencerapparatus, such as a fluorescence DNA sequencer apparatus and the like.Polymerases used for the dideoxy method include, for example, a Klenowfragment of DNA polymerase, AMV reverse transcriptase, Taq DNApolymerase, T7 DNA polymerase, modified T7 DNA polymerase and the like.

[0130] The DNA fragments obtained in the present invention can beincorporated into an appropriate vector, e.g., plasmid pEX, pMAMneo,pKG5, pET3a (Stratagene) and the like as described in detail below, andcan be expressed in appropriate host cells, e.g., E. coli, yeast, CHOcells, COS cells and the like as described in detail below. The DNAfragments can be introduced into animal cells intact, as DNA fragmentswith the addition of an appropriate control sequence or by beingincorporated into an appropriate vector. Transgenic animals whichexpress the given gene can be produced. The animals include mammaliananimals, and include, for example, mice, rats, rabbits, guinea pigs,cattle etc. Preferably, the transgenic animal can be produced byintroducing the DNA fragments into fertilized eggs of an animal such asa mouse. The methods can be carried out according to methods known bythose skilled in the art, for example, the methods described in U.S.Pat. Nos. 4,736,866 and 4,870,009.

[0131] Identification of the given gene product-can be carried out usinganimal cells including cells derived from tumors such as COS1 cells,HT1080 cells and the like suitable therefor into which the gene has beentransfected. The methods where the foreign gene is introduced intoanimal cells of mammals can be carried out by methods known in the artor methods substantially the same thereto, and include, for example, thecalcium phosphate method (e.g., Graham, F. L. et al., Virology, 52:456,1973, etc.), the DEAE-dextran method (e.g., Warden, D. et al., GeneVirol., 3:371, 1968, etc.), the electroporation method (e.g., Neumann,E. et al., EMBO J., 1: 841, 1982, etc.), the microinjection method, theliposome method, the viral infection method, the phage particle methodand the like. The gene product produced by the animal cells transfectedwith the given gene in such ways can also be analyzed.Immunoprecipitation experiments or Western blotting using antibodiessuch as monoclonal antibodies can be used for the analysis.

[0132] Plasmids into which the given gene is incorporated are any ofthose which can express the DNA in the host cells usually used in geneengineering (e.g., prokaryotic hosts such as E. coli, Bacillus subtilis,etc., eukaryotic hosts such as yeast, CHO cells, COS cells etc., insectcell hosts such s Sf21 etc.) In such sequences, for example, thesequence can be modified into codons suitable for expressing in theselected host cells, restriction enzyme sites can be inserted, thesequences can be contained which regulate linkers, adapters serving tobind the target gene, and antibiotic resistance, which regulatemetabolism, and which are useful for selection.

[0133] Preferably, appropriate promoters such as the tryptophan promoter(trp), lactose promoter (lac), tryptophan-lactose promoter (tac),lipoprotein promoter (lpp), λ phage P_(L) promoter and the like can beused for plasmids of which hosts are E. coli, promoters such as SV40late promoter, MMTV LTR promoter, RSV LTR promoter, CMV promoter, SRαpromoter and the like can be used for the plasmids of which hosts areanimal cells, and promoters such as GALL and GAL10 promoters and thelike can be used for the plasmids of which hosts are yeast. Moreover,regulation systems such as CYC1, HIS3, ADH1, PGK, PHO5, GAPDH, ADC1,TRP1, URA3, LEU2, EN0, TP1, AOX1 and the like can also be used.

[0134] An enhancer can be inserted into the vector to facilitate thetranscription of DNA encoding the desired polypeptide, and suchenhancers include those of elements having an action to facilitate thetranscription acting on the promoter and typically having a cis actionof approximately 10 to 100 bp. Many enhancers have been known inmammalian genes such as globin, elastase, albumin, α-fetoprotein,insulin genes and the like. Representatively, the enhancers obtainedfrom eukaryotic infectious viruses can be suitably used, and include,for example, an SV40 enhancer located in the late region of thereplication origin (100-270 bp), an enhancer of the early promoter ofcytomegalovirus, enhancer located in the late region of the replicationorigin in polyoma, an enhancer of adenovirus and the like. A signalsequence fitting for the host can be added if necessary, and those knownby those skilled in the art can be used.

[0135] The plasmids of which hosts are E. coli include, for example,pBR322, pUC18, pUC19, pUC118, pUC119, pSP64, pSP65, pTZ-18R/18U,pTZ-19R/19U, pGEM-3, pGEM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf(−), pBluescriptKS™ (Stratagene) and the like. The plasmid vectors suitable for theexpression in E. coli also include pAS, pKK223 (Pharmacia), pMC1403,pMC931, pKC30, PRSET-B (Invitrogen) and the like. The plasmids of whichhosts are animal cells include the SV40 vector, polyoma viral vector,vaccinia viral vector, retroviral vector and the like, and include, forexample, pcD, pcD-SRα, CDM8, pCEV4, pME18S, pBC12BI, pSG5 (Stratagene)and the like. The plasmids of which hosts are yeast include YIp, YEp,YRp, YCp type vectors and the like, and, for example, pGPD-2 and thelike. The host cells which are E. coli include those derived from the E.coli K12 strain and, for example, NM533, XL1-Blue, C600, DH1, DH5,DH11S, DH12S, DH5α, DH10B, HB101, MC1061, JM109, STBL2, BL21(DE3)/pLYsSand the like. The host cells which are yeast include, for example,Saccharomyces cerevisiae, Schizosaccharomyces prombe, Pichia pastoris,Kluyveromyces strains, Candida, Trichoderma reesia and the other yeaststrains. The host cells which are animal cells include, for example,COS-7 cells, COS-1 cells, CV-1 cells derived from fibroblasts of Africangrivet, 293 cells derived from human renal cells, A431 cells derivedfrom human epidermal cells, 205 cells derived from human colon cells,COP cells, MOP cells, WOP cells derived from murine fibroblasts, CHOcells, CHO DHFR cells derived from Chinese hamster cells, human HeLacells, C127 cells derived from murine cells, NIH3T3 cells derived frommurine cells, murine L cells, 9BHK, HL-60, U937, HaK and Jurkat cells,other cell lines obtained by transformation, normal diploid cells, celllines induced from primary cultured tissue in vitro, and the like.Insect cells include cells using Spodoptera frugiperda (caterpillar),Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophilamelanogaster (fruitfly), silk worm larva or cultured cells, e.g., BM-Ncells using silk worm (Bombyx mori nuclear polyhedrosis virus) or thosederived therefrom or other suitable ones as the vector (for example,Luckow et al., Bio/Technology, 6:47-55, 1988; Setlow, J. K. et al., ed.,Genetic Engineering, Vol. 8, pp. 277-279, Plenum Publishing, 1986; Maedaet al., Nature, 315:592-594, 1985). Utilizing Agrobacterium tumefaciensetc., it is possible to use plant cells as the host cells, which havebeen widely known along with vectors suitable therefor in the art.

[0136] In the gene engineering techniques of the present invention, itis possible to use restriction enzymes, reverse transcriptases known andwidely used in the art, DNA modification enzymes, DNase, DNApolymerases, terminal nucleotidyltransferases, DNA ligases and the liketo modify or convert DNA into a structure suitable for cloning the DNAfragments. For example, restriction enzymes include those described in,for example, Roberts, R. J., Nucleic Acids Res., 13: r165, 1985; Linn,S. et al., ed., Nucleases, p.109, Cold Spring Harbor Lab., Cold SpringHarbor, N.Y., 1982; Roberts, R. J., Macelis, D., Nucleic Acids Res.,19:Suppl. 2077, 1991 and the like. Reverse transcriptases include, forexample, reverse transcriptases derived from mouse Moloney leukemiavirus (MMLV), avian myeloblastosis virus (AMV), and the like. As thereverse transcriptase, the RNase H defective is preferably used,especially, the modified MMLV RT defective for RNase H is preferablyused, and those with heat stability are preferable. The suitable reversetranscriptases include MMLV RT (Gibco-BRL), Superscript RT plus (LifeTechnologies), and the like.

[0137] DNA polymerases include, for example, E. coli DNA polymerase, theKlenow fragment which is a derivative thereof, E. coli phage T4 DNApolymerase, E. coli phage T7 DNA polymerase, heat resistant bacterialDNA polymerase and the like. Terminal nucleotidyltransferases include,for example, TdTase which adds deoxyribonucleotide (dNMP) to 3′-OHterminus described in Wu, R. et al., “Methods in Enzymology” Vol. 100,p96, Academic Press, New York, 1983, and the like. DNA modificationenzymes and DNases include exonucleases and endonucleases and the like,and include, for example, snake venom phosphodiesterase, splenicphosphodiesterase, E. coli DNA exonuclease I, E. coli DNA exonucleaseIII, E. coli DNA exonuclease VII, λ exonuclease, DNase I, nuclease S1,micrococcus nuclease and the like. DNA ligases include, for example, E.coli DNA ligase, T4 DNA ligase and the like. Vectors suitable forconstructing DNA libraries by cloning DNA genes include plasmids, λphages, cosmids, P1 phage, F element, YAC and the like, and includepreferably vectors derived from λ phages, and, for example, Charon 4A,Charon 21A, λgt10, λ gt11, λ DASHII, λ FIXII, λ EMBL3, A ZAPII™(Stratagene) and the like.

[0138] Appropriate selected markers are used, if necessary, for thetransformed cells transformed with the expression vector containing thenucleic acids encoding the protein of the present invention. A cell linestably having a high expression ability can be obtained by cloningrepeatedly. For instance, when a dhfr gene is utilized as a selectedmarker in the transformed cell using animal cells as the host cells,cell lines with higher expression can be obtained by amplifying the DNAencoding the protein of the present invention by culturing with agradual increase in MTX concentration and selecting the resistant celllines. The transformed cells of the present invention can be culturedunder a condition where the nucleic acids encoding the protein of thepresent invention can be expressed, and can produce and accumulate thetarget. The transformed cells can be cultured in media widely used inthe art. For instance, the transformed cells of which hosts areprokaryotic cells such as E. coli and Bacillus subtilis, or yeast cellscan suitably used with liquid media. The media contain carbon sources,nitrogen sources, minerals, and others essential for growth of thetransformed cells. The carbon sources include, for example, glucose,dextrin, soluble starch, sucrose and the like, the nitrogen sourcesinclude, for example, inorganic or organic materials such as ammoniumsalts, nitrates, cornsteep, liquor, peptone, casein, meat extract, wheatgerm extract, soy bean bran, potato extract solution and the like, andthe inorganic materials include, for example, potassium chloride,potassium dihydrogen phosphate, magnesium chloride, calcium carbonateand the like. Also, yeast, vitamins, casamino acids, growth factors andthe like may be added. Also, reagents such as 3β-indolyl acrylate can beadded, if necessary, to operate the promoter efficiently. The range ofpH in the media is preferably about 5 to 8.

[0139] The culture of, for example, E. coli is typically carried out atabout 15 to 45° C. for about 3 to 75 hrs, and ventilation and stirringcan be added if necessary. When the transformed cells of which the hostsare animal cells are cultured, for example, MEM medium, PRMI 1640medium, DMEM medium and the like containing about 5 to 20% fetal calfserum are used as the medium. The range of pH is preferably about 6 to8. The culture is typically carried out at about 30 to 40° C. for about15 to 72 hrs, and ventilation and stirring are added if necessary. Theobtained cells and media can be used just as they are.

[0140] When the desired product is extracted from the above culturedcells, methods can be appropriately used in which the bacteria or cellsare collected by methods known in the art after the culture, suspendedin an appropriate buffer followed by being disrupted by sonication,lysozyme and/or freezing/thawing and the like, and subsequently thecrude extract solution is obtained by centrifugation or filtration.Protein denaturants such as urea, guanidine hydrochloride and the likeand surfactants such as Triton X-100 (trade name), Tween-80 (trade name)and the like may be added in the buffer. When the target product issecreted in the culture medium, a supernatant is separated from thebacteria or cells after completion of the culture by methods known inthe art, and the supernatant is collected. The target product containedin the culture supernatant or the extract solution obtained in such wayscan be purified by appropriately combining the isolation andpurification methods known in the art. For instance, the product can beobtained by purifying by the methods such as salting out ammoniumsulfate precipitation, the gel filtration methods with Sephadex and thelike, the ion exchanging chromatography methods using carriers such asdiethylaminoethyl groups or carboxymethyl groups and the like, thehydrophobic chromatography methods using carriers having hydrophobicgroups such as butyl, octyl, phenyl groups and the like, the dye gelchromatography methods, electrophoresis, dialysis, ultrafiltration, theaffinity chromatography methods, the high performance liquidchromatography methods, and the like. Preferably, the product can beisolated and purified by treating by the polyacrylamide gelelectrophoresis, the affinity chromatography immobilized antibodies suchas monoclonal antibodies which react specifically with antigens, and thelike. For example, included are gelatin-agarose affinity chromatography,heparin-agarose chromatography and the like.

[0141] According to the present invention, provided is a screeningmethod for substances which inhibit formation of the complex formed ofmultiple MT1-MMP, e.g., the formation of homodimer of MT1-MMP, using asystem in which MT1-MMP and proMMP-2 co-exist. For instance, substanceswhich inhibit the formation of homodimer of MT1-MMP can be screenedusing a chimera molecule (including the transformed cells which expressthe molecule or the culture thereof) in which TM/CP of MT1-MMP issubstituted with TM/CP of NGFR.

[0142] In the screening, for example, using the transformed cells whichexpress the chimera molecule in which TM/CP of MT1-MMP is substitutedwith TM/CP of NGFR, or the culture thereof, phosphorylation of thechimera molecule is compared between (i) in the absence of the testsample and (ii) in the case of contacting with the test sample. Inaddition, in the above screening, the biological activities (forexample, protease activity, activation of proMMP-2 induced by thehomodimer formation of MT1-MMP, or cellular migration, invasion and/ormetastasis attributed thereto) can be determined and compared.

[0143] The chimera molecules can be used in intact, or by labeling withfluorescence such as fluorescein, enzymes, radioisotopes and the like.Those which can be preferably used as appropriate tags are added usingthe DNA recombination techniques or labels are added by chemicaltechniques. The determination of the phosphorylation can be carried outby methods known in the art, which include the method detecting thephosphorylation of tyrosine residues induced at the site of TM/CP ofNGFR, with anti-phosphotyrosine antibody and the like. The test samplesinclude, for example, proteins, peptides, non-peptide compounds,synthetic compounds, fermented products, plant extracts, tissue extractsfrom animals, cellular extracts and the like. Examples of the testcompounds used for the test sample may include preferably anti-MMPantibodies, MMP inhibitors, compounds and especially synthetic compoundshaving the inhibitory activity against MMP family members. Inparticular, preferably included are anti-PEX antibodies, pseudo-PEXcompounds and the like. These compounds may be novel compounds orcompounds known in the art. Screening can be carried out according todetermination methods known in the art. Also, it can be done usingvarious markers, buffer systems and other appropriate reagents and thelike, or according to operations as described in the determinationmethods using the following antibodies. The measurement can be typicallycarried out in the buffer such as Tris-HCl buffer, phosphate buffer andthe like which has no adverse effect, for example from pH4 to 10(preferably about pH6 to 8).

[0144] In such respective screening methods, the determination systemsmay be constructed which are associated with the MT1-MMP complexformation of the present invention, by adding normal technicalconsideration of those skilled in the art to typical conditions and theoperating method in the respective methods. Reviews and books can becited for details of these general technical means [for example, seeMethods in Enzymology, Vols. 1, 2, 5 & 6 (Preparation and Assay ofEnzymes); ibid., Vol. 3 (Preparation and Assay of Substrates); ibid.,Vol. 4 (Special Techniques for the Emzymologist); ibid., Vol. 19(Proteolytic Enzymes); ibid., Vol. 45 (Proteolytic Enzymes, Part B);ibid.,. Vol. 80 (Proteolytic Enzymes, Part C), all published fromAcademic Press USA].

[0145] As used herein, the term “antibody” is used in the broadest senseand may cover a single species of desirable monoclonal antibodiesagainst PEX domain protein fragments and antibody compositions having aspecificity to various epitopes thereof, further monovalent orpolyvalent antibodies and polyclonal and monoclonal antibodies, and alsothose which are intact molecules or fragments and derivatives thereof,including F(ab′)₂, Fab′ and Fab fragments, and also chimeric antibodies,hybrid antibodies each having at least two antigen or epitope bindingsites, or bispecific recombinant antibodies (e.g., quadromes, triomes,etc.), interspecies hybrid antibodies, anti-idiotypic antibodies andthose which have been chemically modified or treated and must beregarded as derivatives of these antibodies and further which may beproduced either by adopting cell fusion or hybridoma techniques orantibody engineering or by using synthetical or semisyntheticaltechniques in known manner, which may be prepared either by the knownconventional methods in view of antibody production or by recombinantDNA techniques, and which have neutralizing or binding properties withrespect to the target antigen substances or target epitopes describedand defined herein.

[0146] Monoclonal antibodies prepared against antigenic substances areproduced by any method capable of providing production of antibodymolecules by a series of cell lines in culture. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. The individual antibodies are those containing a population ofidentical antibodies except for possible naturally occurring mutationsthat may be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. In contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The monoclonal antibodiesincluded within the scope of the invention include hybrid andrecombinant antibodies. They are obtainable by substituting a constantdomain of an antibody for a variable domain (e.g., “humanized”antibodies), or a heavy chain for a light chain, by substituting a chainfrom one species with a chain from another species, or by fusing toheterogeneous proteins, regardless of species of origin orimmunoglobulin class or subclass designation, so long as they exhibitthe desired biological activity (e.g., U.S. Pat. No. 4,816,567;Monoclonal Antibody Production Techniques and Applications, pp.79-97,Marcel Dekker, Inc., New York, 1987; etc.).

[0147] Preferable techniques for producing monoclonal antibodiesinclude, for example, the methods using hybridoma cells (G. Kohler andC. Milstein, Nature, 256: 495-497, 1975); the methods using human B cellhybridomas (Kozbor et al., Immunology Today, 4: 72-79, 1983; Kozbor, J.Immunol., 133: 3001, 1984; Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp.51-63, Marcel Dekker, Inc.,New York (1987); triome methods; EBV-hybridoma methods (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96(1985))(techniques for production of human monoclonal antibodies); U.S.Pat. No. 4,946,778 (techniques for production of single-chainantibodies), as well as the following documents:

[0148] S. Biocca et al., EMBO J, 9: 101-108, 1990; R. E. Bird et al.,Science, 242: 423-426, 1988; M. A. Boss et al., Nucl. Acids Res., 12:3791-3806, 1984; J. Bukovsky et al., Hybridoma, 6: 219-228, 1987; M.DAINO et al., Anal. Biochem., 166: 223-229, 1987; J. S. Huston et al.,Proc. Natl. Acad. Sci. USA, 85; 5879-5883, 1988; P. T. Jones et al.,Nature, 321: 522-525, 1986; J. J. Langone et al. (ed.), “Methods inEnzymology”, Vol. 121 (Immunochemical Techniques, Part I: HybridomaTechnology and Monoclonal Antibodies), Academic Press, New York (1986);S. Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855, 1984; V.T. Oi et al., BioTechniques, 4: 214-221, 1986; L. Riechmann et al.,Nature, 332: 323-327, 1988; A. Tramontano et al., Proc. Natl. Acad. Sci.USA, 83: 6736-6740, 1986; C. Wood et al., Nature, 314: 446-449, 1985;Nature, 314: 452-454, 1985, or documents quoted therein (the disclosuresof which are incorporated herein by reference).

[0149] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey have the desirable biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855, 1984).

[0150] Described herein below is the production of antibodies, includingembodiments of monoclonal antibodies.

[0151] It goes without saying that the monoclonal antibody to be used inthe present invention may be a monoclonal antibody obtained by utilizingcell fusion techniques with myeloma cells.

[0152] The monoclonal antibody to be used in the present invention canbe produced by the following processes:

[0153] 1. Preparation of immunogenic antigens (immunogens)

[0154] 2. Immunization of animals with immunogenic antigens

[0155] 3. Preparation of myeloma cells

[0156] 4. Cell fusion between antibody-producing cells and myeloma cells

[0157] 5. Selection and cloning of hybridomas (hybrid cells)

[0158] 6. Production of monoclonal antibodies

[0159] 1. Preparation of Immunogenic Antigens

[0160] The antigen as used herein includes isolated MT1-MMP PEX proteinfragments and derivatives thereof, produced, as disclosed above, andsuitable synthetic oligopeptides, chemically synthesized, based oninformation on amino acid sequences for the PEX domain of MT1-MMPsequenced (GenBank™/EMBL accession number: D26512). Although the antigenmay be used to immunize animals after being mixed with a suitableadjuvant without any modifications, it can be used after formation ofimmunogenic conjugates. The antigen for such an immunogen may beselected from at least three consecutive amino acid residues from thePEX domain of MT1-MMP (e.g., the region ranging from amino acid residue316 to acid residue 508 among the MT1-MMP amino acid sequence, or theregion from amino acid residue 319 to amino acid residue 535 among theMT1-MMP amino acid sequence in some cases). For instance, the antigensmay be a fragment derived from MT1-MMP PEX, or a synthetic polypeptidefragment obtained via selecting characteristic sequence areas based onthe amino acid sequences of MT1-MMP PEX areas followed by design andchemical synthesis. The fragments may be coupled with various carrierproteins via suitable coupling agents to form immunogenic conjugatessuch as hapten-proteins. The immunogenic conjugates can be used todesign monoclonal antibodies that can react with (or recognize) specificsequences exclusively. A cysteine residue or the like can be added tothe polypeptide thus designed so as to prepare an immunogenic conjugateeasily. To couple with a carrier protein or the like, the carrierprotein is first activated. This activation may include incorporation ofan activated binding group thereinto, etc. The activated binding groupsinclude (1) active ester or active carboxyl groups such as a nitrophenylester group, a pentafluorophenyl ester group, a 1-benzotriazol estergroup, and an N-succinimido ester group; (2) active dithio groups suchas a 2-pyridyldithio group, etc. The carrier proteins include keyholelimpet haemocyanin (KLH), bovine serum albumin (BSA), ovalbumin,globulin, polypeptides such as polylysine, bacterial components such asBCG or the like.

[0161] 2. Immunization of Animals With Immunogenic Antigens

[0162] Animals can be immunized according to techniques as described inShigeru MURAMATSU et al. ed., “Jikken Seibutsu Gaku Kouza 14, Men-ekiSeibutsu Gaku (Lectures on Experimental Biology 14, Immunobiology)”,Maruzen K. K., 1985; Nippon Seikagaku Kai (Biochemical Society of Japan)ed., “Zoku-Seikagaku Jikken Kouza 5, Men-eki Seikagaku Kenkyuho(Lectures on Biochemical Experiments (Second Series; 5), Methods forImmunological and Biochemical Study)”, Tokyo Kagaku Dojin, Japan (1986);Nippon Seikagaku Kai (Biochemical Society of Japan) ed., “Shin-SeikagakuJikken Kouza 12, Bunshi Men-eki Gaku III (Kougen-Koutai-Hotai) (NewLectures on Biochemical Experiments 12, Molecular Immunology III(Antigen-Antibody-Complement))”, Tokyo Kagaku Dojin, Japan (1992); etc.,the disclosures of which are hereby incorporated by reference.Immunization can be performed in a mammal, for example, by one or moreinjections of an immunizing agent (and, if desired, an adjuvant).Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the aforementioned antigen peptides orrecombinant MT1-MMP proteins or recombinant MT1-MMP PEX obtained by DNAcloning or their fragments obtained by enzymatic digestion thereof. Itmay be useful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins which may be employed include the aforementioned carrierproteins. The adjuvant to be used with the antigen includes Freund'scomplete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, liposome,aluminium hydroxide, silica, lipid A, synthetic trehalosedicorynomycolate (TDM) adjuvant, etc.

[0163] The Immunization is carried out with suitable animals, includingmice such as BALB/c, hamsters, and others. The antigen dose is, forexample, approximately 1 to 400 μg/animal for mice. Generally, theantigen is injected intraperitoneally or subcutaneously into a hostanimal, followed by additional immunization by repeated courses whereinintraperitoneal, subcutaneous or intravenous administrations are carriedout approximately 2 to 10 times at 1- to 4-week intervals, preferably 1-to 2-week intervals. For immunization, BALB/c mice, as well as F1 micebetween BALB/c mice and other mice, etc. can be used. As required, thelevels of animal immunization can be assessed by constructing a systemfor measuring a titer of antibody and measuring the titer of anantibody. The antibody of the present invention may include thoseobtainable from such immunized animals, for example, anti-serum,polyclonal antibodies, etc.

[0164] 3. Preparation of Myeloma Cells

[0165] Immortal cell strains (tumor cell lines) to be used for cellfusion can be selected from non-immunoglobulin-producing cell lines. Thecell strains to be used for cell fusion may include, for example,P3-NS-1-Ag4-1 (NS-1, Eur. J. Immunol., 6: 511-519, 1976), SP-2/0-Ag14(SP-2, Nature, 276: 269-270, 1978), mouse myeloma MOPC-21 cellline-derived P3-X63-Ag8-U1 (P3U1, Current topics Microbiol. Immunol.,81: 1-7, 1978), P3-X63-Ag8 (X63, Nature, 256: 495-497, 1975),P3-X63-Ag8-653 (653, J. Immunol., 123: 1548-1550, 1979), etc.8-Azaguanine resistant mouse myeloma cell lines can be sub-cultured in amedium for cell culture, such as Dulbecco's modified Eagle's medium(DMEM) or RPMI-1640 medium, supplemented with antibiotics such aspenicillin, amikacin or the like, fatal calf serum (FCS) or the like and8-azaguanine (for example, 5 to 45 μg/ml). The specified number of celllines can be prepared by passage the normal medium two or five daysbefore cell fusion. The cell lines to be used may be cultured on thenormal medium after the frozen and preserved strains have beencompletely thawed at approximately 37° C. and have been washed on thenormal medium such as RPMI-1640 three or more times, and the specifiednumber of cell strains may be prepared.

[0166] 4. Cell Fusion Between Antibody-Producing Cells and Myeloma Cells

[0167] After animals such as mice are immunized according to the abovestep 2, their spleens are taken out in two to five days from finalimmunization, and the spleen cell suspension is obtained. In addition tothe spleen cells, lymph node cells at various sites of organisms can beobtained and used for cell fusion. The spleen cell suspension thusobtained and the myeloma cell strains obtained by the above step 3 areplaced in a medium such as minimum essential medium (MEM), DMEM andRPMI-1640 medium, followed by addition of an agent for cell fusion, suchas polyethylene glycol. A widely-used agent for cell fusion can be used,including inactivated HVJ (Hemagglutinating virus of Japan, “Sendaivirus”) and the like. Preferably, 0.5 to 2 ml of 30 to 60% polyethyleneglycol can be added. Polyethylene glycol with molecular weights from1,000 to 8,000 can be employed, more preferably, polyethylene glycolwith molecular weights from 1,000 to 4,000. The preferred concentrationof polyethylene glycol in the fusion medium is from 30 to 60%. Asrequired, a small amount of dimethyl sulfoxide or the like is added topromote fusion. The ratio of spleen cells (lymphocytes): myeloma celllines to be used for fusion is preferably 1:1 to 20:1, and preferablyfalls within 4:1 to 7:1. The fusion reaction is conducted for 1 to 10min, before the addition of a medium such as RPMI-1640 medium. Fusionreaction can be done several times. After fusion reaction, cells areseparated by a centrifuge, then transferred to the selection medium.

[0168] 5. Selection and Cloning of Hybridomas (Hybrid Cells)

[0169] The selection media include conventionally known “HAT medium”,i.e., FCS-containing MEM, RPMI-1640 medium, etc., supplemented withhypoxanthine, aminopterin, and thymidine. The replacement method for theselection medium is to replenish an amount equivalent to the capacitydispensed to the medium plate on the following day, after which themedium is replaced by half an amount in HAT medium every one to threedays. The replacement can be modified depending on situations. Eight tosixteen days after fusion, the medium may be replaced every one to fourdays with conventionally known “HT medium” wherein aminopterin isexcluded. As a feeder cell, for example, mouse thymocyte can be used,which is sometimes effective.

[0170] The supernatant of the culture well with vigorously growinghybridoma is screened by using a predetermined peptide fragment as anantigen or by using a labeled anti-mouse antibody for measuring targetantibodies, with a measuring system such as radioimmunoassay (RIA),enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay(FIA) or by the fluorescence activated cell sorter (FACS), etc. Thetarget antibody-producing hybridoma is cloned. Cloning is carried out bypicking up colonies in the agar medium or by the limiting dilution. Thelimiting dilution is preferred. Cloning should be performed severaltimes.

[0171] 6. Production of Monoclonal Antibodies

[0172] The obtained hybridoma cells are cultured in a suitable growthmedium such as FCS-containing MEM, RPMI-1640 medium or the like, and adesired monoclonal antibody can be obtained from the culturesupernatant. Large amounts of monoclonal antibodies can be produced bypropagating hybridomas as ascites tumors, etc. In this case, eachhybridoma is implanted intraperitoneally in a histocompatible animalisogenic to an animal from which the myeloma cell is derived and ispropagated. Or each hybridoma can be inoculated, for example, in nudemice, and propagated to produce the monoclonal antibody in the ascitesof the animals. The produced monoclonal antibody can be collected fromthe ascetic fluid and obtained. Prior to implantation of hybridomas, theanimal is pretreated intraperitoneally with mineral oils such asPristane (2,6,10,14-tetramethylpentadecane). After the pretreatment, thehybridoma can be propagated therein and the ascitic fluid can beharvested. The ascitic fluid can be used as a monoclonal antibodywithout purification or after purification by conventionally knownmethods, including salting out such as precipitation with ammoniumsulfate, gel filtration with Sephadex and the like, ion exchangechromatography, electrophoresis, dialysis, ultrafiltration, affinitychromatography, high-performance liquid chromatography, etc. Theisolated or purified products can be employed as monoclonal antibodies.Preferably, the monoclonal antibody-containing ascitic fluid isfractionated with ammonium sulfate and separated and purified bytreatments with anion exchange gel such as DEAE-Sepharose, an affinitycolumn such as protein A column, etc. More preferably, it is treatedwith affinity chromatography using immobilized antigens or antigenfragments (for example, synthetic peptides, recombinant antigen proteinsor peptides, portions which the antibody can specifically recognize,etc.); affinity chromatography with immobilized protein A;hydroxyapatite chromatography; etc.

[0173] In addition, it is possible to use transgenic mice and otherorganisms including other mammals for expressing antibodies such ashumanized antibodies against the immunogenic polypeptide products of thepresent invention.

[0174] It is also possible to produce antibodies with recombinant DNAtechniques wherein the antibody thus obtained in a large amount issequenced and/or a nucleotide sequence coding for the antibody obtainedfrom the hybridoma cell line is employed.

[0175] DNA coding for the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy or light chain of murine antibodies). Once isolated, the DNA maybe placed, according to the aforementioned techniques, into expressionvectors, which are then transfected into host cells such as CHO cells orCOS cells. The DNA may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains forthe homologous murine sequences (Morrison et al., Proc. Natl. Acad. Sci.USA, 81: 6581, 1984). Thus, chimeric and hybrid antibodies having adesired binding specificity can be prepared. Further, antibodies can bemodified, including preparations of chimeric or hybrid antibodies byadaptations of known methods in synthetic protein chemistry, includingthose involving coupling agents as listed hereinbelow.

[0176] Humanized antibodies are achievable by known techniques in theart (e.g., Jones et al., Nature, 321: 522-525, 1986; Riechmann et al.,Nature, 332: 323-327, 1988; Verhoeyen et al., Science, 239: 1534-1536,1988).

[0177] Human monoclonal antibodies can be achieved according to knowntechniques in the art. For producing human monoclonal antibodies, humanmyeloma cells and human-mouse hetero-myeloma cells are known in the art(Kozbor, J. Immunol., 133: 3001, 1984; Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp 51-63, MarcelDekker, Inc., New York (1987)). Methods for making bispecific antibodiesare known in the art (Millstein et al., Nature, 305: 537-539, 1983; WO,A, 93/08829; Traunecker et al., EMBO J., 10: 3655-3659, 1991; Suresh etal., “Methods in Enzymology”, Vol. 121, p 210 (1986)).

[0178] These antibodies may be treated with enzymes such as trypsin,papain, pepsin and others and occasionally be subjected to reduction toproduce antibody fragments including Fab, Fab′, and F(ab′)₂. Theseantibody fragments may be occasionally used.

[0179] When employed as pharmaceutical agents, the active components ofthe present invention (e.g., (a) PEX protein of MT1-MMP (MT1PEX),peptide fragments and salts thereof, or mutants, analogs, derivativesthereof, (b) nucleic acids (including DNA, etc.) each encoding the PEXdomain of MT1-MMP, (c) the antibodies (including monoclonal antibodies)of the present invention or derivatives thereof, and (d) compounds, orsalts thereof, which suppress or inhibit a biological activity such ashomodimer formation of MT1-MMP, etc,) may be administered usually in theform of a pharmaceutical composition or preparation alone or inadmixture with a variety of pharmaceutically acceptable aids. Forexample, MT1PEX or a salt thereof can be administered as such forms.Preferably they may be administered in the form of a convenientpharmaceutical composition or formulation suitable for oral, topical,parenteral application, or the like. Any of dosage forms (includingthose for inhalation and rectal administration) may be selecteddepending on purpose.

[0180] The active components of the present invention can also be usedin admixture with anti-tumor agents (antineoplastic agents) and/ortumor-metastasis inhibitors. Any of the anti-tumor agents(antineoplastic agents) and/or tumor-metastasis inhibitors can be usedwithout any limitation so long as they advantageously serve. Examples ofsuch drugs are selected from drugs known in the art.

[0181] The parenteral administration includes topical, percutaneous,intravenous, intramuscular, subcutaneous, intracutaneous, andintraperitoneal routes. It is also possible to apply the drug directlyto affected sites, and, in a certain case, the direct application issuitable. Preferably mammals including human can receive the drug orallyor parenterally (e.g., intracellularly, intra-tissularly, intravenously,intramuscularly, subcutaneously, intracutaneously, intraperitoneally,intrapleurally, intraspinally, by instillation, enterally, per rectum,by instillation into the ear, eye, or nose, by swabbing or applicationon the teeth, skin or mucosa, etc.). Specific dosage forms of thepharmaceutical preparations and formulations include pharmaceuticalsolutions, pharmaceutical dispersions, semisolid preparations,particulate preparations, shaped preparations, extractives, etc.Examples of the dosage forms are tablets, coated tablets, sugar coatedtablets, pills, troches, hard capsules, soft capsules, microcapsules,implants, powders, pulvises, granules, fine granules, injections,liquids and solutions, elixirs, emulsions, irrigations, syrups, aqueousmixtures, milks, suspensions, liniments, lotions, aerosols, sprays,inhalations, nebula, ointments, plasters, patches, pastes, cataplasms,creams, oleates, suppositories (e.g., rectal suppositories), tinctures,dermatologic waters, ophthalmic solutions, collunariums, auristillae,paints, transfusions, powders for injection solutions, lyophilizedpreparations, conditioned gels, etc.

[0182] The pharmaceutical compositions can be formulated in accordancewith conventional techniques. For example, the pharmaceuticalcomposition or formulation may comprise at least one of said compounds(active components) of the present invention or a salt alone or inadmixture with physiologically allowable carriers, pharmaceuticallyacceptable carriers, adjuvants, vehicles, excipients, diluents, etc. Thecompound (active component) of the present invention or a salt thereofis usually admixed with a single member selected from the groupconsisting of physiologically allowable carriers, pharmaceuticallyacceptable carriers, adjuvants, vehicles, excipients, diluents,flavoring agents, perfuming agents, sweetening agents, expanders,antiseptics, stabilizers, binders, pH regulators, buffering agents,detergents (surfactants), bases, solvents, fillers, bulking agents,solution adjuvants, solubilizers, isotonizing agents, emulsifiers,suspending agents, dispersers, thickeners, gelling agents, hardeners,absorbents, adhesives, elastomers, plasticizers, disintegrants, aerosolpropellants, preservatives, antioxidants, opacifying agents, humectants,emollients, charge protectors, soothing agents, etc., or suitably in acombination thereof, depending on necessity, to give a unit dose formwhich is required for generally approved pharmaceutical practices.

[0183] Formulations suitable for parenteral routes include asepticsolutions or suspensions containing at least one active component inadmixture with water or other pharmaceutically acceptable media.Examples of such parenteral formulations are injections. Preferredliquid carriers for injection generally include water, saline, dextrosesolution, other related saccharide solutions, ethanol, glycols such aspropylene glycol and polyethylene glycol, etc. For the preparation ofinjections, the active component of the present invention is usuallyadmixed with any of carriers such as distilled water, Ringer's solution,physiological saline, suitable dispersing agents, moistening agents,suspending agents, etc. to form injectable formulations includingsolutions, suspensions, emulsions, etc. by known techniques in the art.

[0184] Examples of aqueous liquids for the injection are a physiologicalsaline and isotonic solutions containing glucose and other aids (e.g.,D-sorbitol, D-mannitol, sodium chloride, etc.), etc. The aqueous liquidmay be used in admixture with a suitable pharmaceutically acceptablesolubilizing aid such as alcohol (e.g., ethanol, etc.), polyalcohol(e.g., propylene glycol, polyethylene glycol, etc.), nonionicsurface-active agent (e.g., Polysorbate 80™, HCO-50, etc.), etc. Theinjectable oily liquids may include sesame oil, soybean oil, etc. Suchoily liquids may be used in admixture with solubilizing aids such asbenzyl benzoate, and benzyl alcohol. They may also be admixed withbuffers (e.g., phosphate buffer, sodium acetate buffer, etc.) or agentsfor osmoregulation, analgesic agents (e.g., benzalkonium chloride,procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin,polyethylene glycol, etc.), preservatives (e.g., benzyl alcohol, phenol,etc.), antioxidants such as ascorbic acid, absorbefacients, etc. Theprepared injection solution is usually filled in suitable ampoules.

[0185] For parenteral administration, solution or suspension unit dosageforms are prepared in pharmaceutically acceptable sterile fluids such aswater, ethanol, and oils, in admixture with or without detergent andother pharmaceutically acceptable aids. The oily vehicle and solventused in the parenteral formulation may include natural, synthetic orsemi-synthetic mono-, di-, or triglycerides; natural, semi-synthetic orsynthetic fats and oils; and fatty acids. Examples of such oily vehiclesand solvents are plant oils such as peanut oil, corn oil, soybean oil,and sesame oil. For example, this injection can usually be prepared toform unit doses each containing approximately from 0.1 to 10 parts ofthe compound of the present invention per 100 parts by weight of thedose composition.

[0186] The formulation suitable for topical use, such as buccal orrectal application, includes mouthwashes and gargles, dentifrices,sprays for cavitas oris, inhalants, ointments (salves), dental fillers,dental coating agents, dental pastes, suppositories, etc. Themouthwashes and other dental preparations are produced by conventionaltechniques, using pharmaceutically acceptable carriers. For the spraysfor cavitas oris and inhalants, the compound of the present inventioncan be applied to teeth, etc. in the form of an aerosol or solution fornebulizers in which it is dissolved alone or in combination withpharmaceutically acceptable inert carriers, or in the form of powdersfor inhalation. The ointments (salves) are prepared by conventionaltechniques, in admixture with conventionally employed pharmaceuticalbases such as ointment bases (white petrolatum, paraffin, olive oil,macrogol 400, macrogol ointment, etc.).

[0187] The pharmaceutical drugs for topical application (includingpainting) to teeth and skin can be prepared in the form of a solution orsuspension utilizing suitably sterilized water or non-aqueous vehicles.The additives used include buffering agents such as sodium bisulfite anddisodium edetate; preservatives including antiseptic, antimicrobial andantifungal agents such as acetic acid, phenylmercuric nitrate,benzalkonium chloride and chlorhexidine; and thickeners such ashypromellose.

[0188] The suppositories can be prepared by conventional techniquesutilizing carriers well known in the art, preferably suitablenon-irritative excipients. The excipients are preferably those which aresolid at room temperature but liquid at rectal temperature wherein suchsubstances melt in the rectum to deliver a drug. Examples of theexcipients are polyethylene glycols, lanolin, cacao butter, fatty acidtriglycerides, etc. The suppositories are ordinarily prepared in theform of compositions containing the compound of the present invention atranges of approximately from 0.1 to 95 wt %. The ingredients, dependingon the vehicle and concentration used, can be either suspended ordissolved in the vehicle. Adjuvants such as local anesthetics,preservatives and buffering agents can be dissolved in the vehicle.

[0189] The formulations suitable for oral application include solidcompositions such as tablets, pills, capsules, powders, granules, andtroches; fluid compositions such as solutions, syrups, and suspensions;etc. In preparing oral formulations, pharmaceutical adjuvants known inthe art are employed. The tablets and pills can be prepared further byenteric coating. When the unit dosage form is a capsule, fluid carrierssuch as fats and oils can be contained in addition to the aforementionedmaterials.

[0190] Further, for the therapeutic and/or prophylactic agents, asaforementioned, containing the nucleic acids (including DNA) of thepresent invention, said nucleic acid can be applied alone, or byligation with suitable vectors used in genetic recombination techniques(or recombinant DNA techniques) including virus-derived vectors such asvectors derived from retrovirus. The nucleic acids (including DNA) ofthe present invention can be administered by conventional knowntechniques, in unmodified forms or in forms formulated in admixture withsuitable aids or physiologically acceptable carriers, for example, topromote transfer into intracellular compartments. The nucleic acids(including DNA) of the present invention can be presented foradministration to humans and animals in pharmaceutical composition orpreparations as aforementioned. For administration of the nucleic acids(including DNA) of the present invention, techniques known as genetherapy can also be applied.

[0191] Dose levels of said active component according to the presentinvention may vary within a wide range. Specific dose levels andadministration cycles for any particular patient will be employeddepending upon a variety of factors including the activity of specificcompounds employed, the sex, age, body weight, general health, diet,time of administration, route of administration, rate of excretion, drugcombination, and the severity of the particular disease undergoingtherapy, or other factors.

[0192] For the manufacture of pharmaceutical compositions andpreparations, the additives, etc., preparation methods and the like canbe suitably selected, depending on necessity, from those disclosed inNippon Yakkyokuho Kaisetsusho Henshu Iinkai (ed.), “13th ed. NipponYakkyokuho Kaisetsusho (Commentary on The Pharmacopoeia of Japan, 13thed.)”, Jul. 10, 1996, Hirokawa Pub. Co., Tokyo, Japan; HisashiIchibagade et al. (ed.), “Pharmaceutical Research and Development (IkuoSuzuki, chief editor), Vol. 12 (Pharmaceutical Necessities 1)”, Oct. 15,1990, Hirokawa Pub. Co., Tokyo, Japan; ibid., Vol. 12 (PharmaceuticalNecessities 2), Oct. 28, 1990, Hirokawa Pub. Co., Tokyo, Japan; etc.,all the disclosures of which are incorporated herein by reference.

[0193] The active components of the present invention are not limited aslong as they serve as suppressors and/or inhibitors against biologicalactivities such as the homodimer formation of MT1-MMP. Preferableexamples of the active components include substances having anadvantageous property. The active components of the present inventioninclude for example, (a) PEX protein of MT1-MMP, fragments or saltsthereof, mutants, analogs or derivatives thereof, (b) nucleic acids suchas DNA etc., encoding the PEX domain of the MT1-MMP, (c) the antibodies(including monoclonal antibodies) of the present invention, partialfragments thereof or derivatives thereof, and (d) the compounds or saltsthereof which suppress and/or inhibit biological activities such as thehomodimer formation of MT1-MMP.

[0194] The active ingredient of the present invention is anticipated tobe useful for suppressing or inhibiting the processing of proMMP-2induced by the formation of the complex formed of multiple MT1-MMP and,for example, at least the process converting ProMMP-2 to the activatedtype by the homodimer formation of MT1-MMP. The active ingredient isuseful for maintenance or recovery of a function which is affectedand/or disappears by excess activation of proMMP-2, and is useful forprevention or treatment of disorders, abnormalities and/or diseases withwhich the processing of proMMP-2 induced by the formation of the complexformed of multiple MT1-MMP, for example, at least the activation ofproMMP-2 by the homodimer formation of MT1-MMP is associated. Also, itis useful for suppressing or inhibiting the processing of proMMP-2 byMT1-MMP. Additionally, it is useful for controlling, for examplesuppressing the migration, invasion and/or metastasis of MT1-MMPgene-expressing cells, and is anticipated to be useful for theprevention or treatment of disorders, abnormalities and/or diseasesinduced by the migration, invasion and/or metastasis of the cells inassociation with the processing of proMMP-2 by MT1-MMP in the expressingcells. Furthermore, the active ingredients including MT1PEX protein andantibodies against PEX of the present invention are useful forcontrolling, for example, suppressing the migration, invasion and/ormetastasis of MT1-MMP gene-expressing cells, and are useful for theprevention or treatment of disorders, abnormalities and/or diseasesinduced by the increased migration, invasion and/or metastasis of thecells in association with the activation of proMMP-2 by the homodimerformation of the MT1-MMP. In particular, they are useful for blockingand/or suppressing the migration, invasion and/or metastasis of tumorcells, such as cancers, and can be anticipated as anti-tumor agentsand/or cancer metastasis suppressors.

[0195] Kits

[0196] The present invention further relates topharmaceutically-acceptable packs (and/or containers or packages) andkits comprising one or more containers filled with one or more of thecomponents of the aforementioned compositions of the invention.Associated with such a single or plural containers can be a notice(attached document) in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, reflecting approval by the agency of the manufacture, use orsale of the product for human administration.

[0197] According to the present invention, the inventive drug design maybe performed utilizing techniques applicable in the art, including forexample the following steps:

[0198] selecting a site or region selected from the group consisting ofthose comprising at least 2 continuous amino acid residues selected fromconstituent amino acid residues for a sequence ranging from amino acidresidues for the PEX domain of MT1-MMP, and next

[0199] (i) substituting an isoster for a pharmacophore among them,

[0200] (ii) replacing at least one of constituent amino acid residueswith a D-amino acid residue,

[0201] (iii) modifying a side chain of amino acid residues,

[0202] (iv) arranging and linking an amino acid residue different fromamino acid residues present in the said sequences, or

[0203] (v) analyzing a stereostructure and designing mimics (e.g.,Koichi Shudo (ed.), “Pharmaceutical Research and Development, Vol. 7(Drug Design)”, Jun. 25, 1990, Hirokawa Pub. Co., Tokyo, Japan anddocuments & articles quoted therein, etc.). Part of such techniquesincludes those which are disclosed herein above.

[0204] For terms (words) and/or abbreviations used in the specificationand in the drawings, they must conform with an “IUPAC-IUB Commission onBiochemical Nomenclature” or are based on the meanings of the termswhich are commonly used in the art. Abbreviations as listed below areprincipally used hereinbelow:

[0205] bp: base pair(s);

[0206] BB-94: [4-(N-hydroxyamino)-2R-isobutyl-3S-(thiophen-2-ylthiomethyl)-succinyl]-L-phenylalanine-N-methylamide or batimastat

[0207] TIMP-2: tissue inhibitor of metalloproteinase-2

[0208] IPTG: isopropyl-1-thio-β-D-galactopyranoside

[0209] PCR: polymerase chain reaction

[0210] SDS: sodium dodecyl sulfate

[0211] For amino acid sequences in relation with proteins, peptides,etc: A: alanine (Ala) C: cysteine (Cys) D: aspartic acid (Asp) E:glutamic acid (Glu) F: phenylalanine (Phe) G: glycine (Gly) H: histidine(His) I: isoleucine (Ile) K: lysine (Lys) L: leucine (Leu) M: methionine(Met) N: asparagine (Asn) P: proline (Pro) Q: glutamine (Gln) R:arginine (Arg) S: serine (Ser) T: threonine (Thr) V: valine (Val) W:tryptophan (Trp) Y: tyrosine (Tyr) For nucleotide sequences: A: adenineC: cytosine G: guanine T: thymine

EXAMPLES

[0212] The present invention is specifically described by means of thefollowing Examples which are provided only for illustrative purposes,and reference to specific embodiments of the present invention. Althoughthese illustrative examples are provided for disclosing particularembodiments of the present invention, they should not be construed aslimiting or restricting the scope of the present invention disclosedherein. It should be understood that various modes will be practicablebased on the spirit of the present invention.

[0213] All the examples were or can be practiced using standardtechniques well or conventionally known to those of ordinary skill inthe art unless otherwise specified.

[0214] Molecular biological techniques generally employed in thefollowing Examples can be carried out as described in a standardexperimental manual, for example, J. Sambrook, E. F. Fritsch & T.Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. All “parts” oramounts presented in the following Examples are according to weightbasis unless defined and referred to in particular.

[0215] Size separation of fragments in the following Examples wascarried out using a procedure described in the literature describedabove or in many other reference literature, e.g., Goeddel et al.,Nucleic Acids Res., 8: 4057, 1980, namely, standard techniques such asagar and polyacrylamide gel electrophoresis (“PAGE”). Unless otherwisedescribed, ligation was carried out with a standard buffer solution,incubation temperature and time, at an approximate amount of equivalentmolar concentration to a DNA fragment to be ligated, using about 10units of T4 DNA ligase (“ligase”) per 0.5 μg of DNA.

[0216] In the following Examples, unless particularly indicated,specific operation and conditions for treatment were according to:

[0217] J. Sambrook, E. F. Fritsch & T. Maniatis (ed.), “MolecularCloning: A Laboratory Manual (2nd ed.)”, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989 and D. M. Glover et al. (ed.),“DNA Cloning”, 2nd ed., Vol. 1 to 4, (The Practical Approach Series),IRL Press, Oxford University Press, 1995 and the like for DNA cloning;

[0218] R. Saiki et al., Science, 230: 1350, 1985; R. Saiki et al.,Science, 239: 487, 1988; H. A. Erlich (ed.), PCR Technology, StocktonPress, 1989; D. M. Glover et al. (ed.), “DNA Cloning”, 2nd ed., Vol. 1,(The Practical Approach Series), IRL Press, Oxford University Press,1995; M. A. Innis et al. (ed.), “PCR Protocols: A Guide to Methods andApplications”, Academic Press, New York, 1990; M. J. McPherson, P.Quirke and G. R. Taylor (ed.), PCR: A Practical Approach, IRL Press,Oxford, 1991; M. A. Frohman et al., Proc. Natl. Acad. Sci. USA, 85:8998-9002, 1988 and the like for PCR in particular;

[0219] G. B. Fields (ed.), “Methods in Enzymology”, Vol. 289(Solid-Phase Peptide Synthesis), Academic Press, New York (1997) and thelike for peptide synthesis. Furthermore, in instances where commerciallyavailable agents or kits were employed, protocols attached thereto orreagents and the like attached thereto were used.

[0220] Moreover, the following procedures were according to additionalliterature described below. Namely, these procedures can be carried outin accordance with a method described in:

[0221] L. Grossman et al. (ed.), “Methods in Enzymology”, Vol. 12 (PartA & Part B: Nucleic Acids), Academic Press, New York, 1968; S. L. Bergeret al. (ed.), “Methods in Enzymology”, Vol. 152 (Guide to MolecularCloning Techniques), Academic Press, New York, 1987; p. 33 & p. 215 andthe like, for example, for isolation of mRNA;

[0222] R. Wu (ed.), “Methods in Enzymology”, Vol. 68 (Recombinant DNA),Academic Press, New York, 1980; R. Wu et al. (ed.), “Methods inEnzymology”, Vol. 100 (Recombinant DNA, Part B) & Vol. 101 (RecombinantDNA, Part C), Academic Press, New York, 1983; R. Wu et al. (ed.),“Methods in Enzymology”, Vol. 153 (Recombinant DNA, Part D) & Vol. 154(Recombinant DNA, Part E), Academic Press, New York, 1987; R. Wu (ed.),“Methods in Enzymology”, Vol. 155 (Recombinant DNA, Part F), AcademicPress, New York, 1987; J. H. Miller (ed.), “Methods in Enzymology”, Vol.204, Academic Press, New York, 1991; R. Wu (ed.), “Methods inEnzymology”, Vol. 216 (Recombinant DNA, Part G), Vol. 217 (RecombinantDNA, Part H) & Vol. 218 (Recombinant DNA, Part I), Academic Press, NewYork, 1993; S. Weissman (ed.), “Methods in Enzymology”, Vol. 303,Academic Press, New York, 1999; J. C. Glorioso et al. (ed.), “Methods inEnzymology”, Vol. 306, Academic Press, New York, 1999; JapaneseBiochemical Society (ed.), “Zoku Seikagaku Jikken Koza 1, IdenshiKenkyu-ho II”, Tokyo Kagaku-dojin Publishing Co., Inc., 1986; JapaneseBiochemical Society (ed.), “Shin-Seikagaku Jikken Koza 2, Kakusan III(Recombinant DNA technique)”, Tokyo Kagaku-dojin Publishing Co., Inc.,1992 and the like, for example, for DNA cloning;

[0223] L. Grossman et al. (ed.), “Methods in Enzymology”, Vol. 29(Nucleic Acids and Protein Synthesis, Part E), Academic Press, New York,1974 and the like, for example, for hybridization. Alternatively, amethod described in literature cited in the above literature or a methodsubstantially equivalent to the same or a modified method thereof can beperformed (Description in literature is incorporated in the disclosureof the present specification as reference.)

[0224] Experimental materials and reagents used in the followingExamples are as described hereinafter.

[0225] Dulbecco's modified Eagle's medium (DMEM) was manufactured byNissui Pharmaceutical Co., Ltd (Tokyo, Japan). FuGENE 6™ wasmanufactured by Roche Molecular Biochemicals Co., Ltd., Basel,Switzerland. A mouse monoclonal anti-FLAG epitope M2 antibody and analkaline phosphatase-conjugated goat anti-mouse IgG antibody weremanufactured by Sigma Chemical Co., Ltd. (MO, USA). Monoclonalanti-phosphotyrosine antibody PY20 was manufactured by ICN Biochemicals,Inc., (Ohio, USA). Chemical crosslinking agent DSG was manufactured byPierce Chemical Co. (1L, USA). DQ™ gelatin was manufactured by MolecularProbes, Inc., (OR, USA). Matrigel Matrix was manufactured by BectonDickinson Labware (MA, USA). Peptidyl hydroxamate MMP inhibitor BB-94was provided by Dr. Peter D. Brown of British Biotech PharmaceuticalsLtd. (Oxford, UK).

Example 1

[0226] A

[0227] (1) Construction of PEX-Deleted FLAG-Tagged MT1-MMP (MT1Cat-F),Catalytic Domain-Deleted FLAG-Tagged MT1-MMP (MT1PEX-F), MT1-F/NGFR,MT1PEX-F/NGFR, MT1Cat-F/NGFR and MT4PEX-F/NGFR

[0228] A FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-tagged MT1-MMP(MT1-F) expression construct was generated as described in Itoh, Y. etal., J. Biol. Chem. 274: 34260-34266, 1999. This construct was used as atemplate to generate cDNA fragments for MT1Cat-F (Δ Ile³¹⁸-Gly⁵³⁵) andMT1-PEX-F (Δ Tyr¹¹²-Pro³¹²) by the PCR extension method of Ho et al.,(Ho, S. N. et al., Gene, 77: 51-59, 1989). The cDNA was subcloned intopSG5 (Stratagene, CA, USA).

[0229] Chimera mutants of the ectodomain of MT1-F, MT1Cat-F, MT1PEX-F orMT4PEX-F, with the transmembrane (TM)/cytoplasmic (CP) domain of NGFRwere also generated by the PCR method in the same manner, and subclonedinto pSG5.

[0230] The mutant is derived from sequences corresponding to Met¹ toAsp⁵¹⁵ of MT1-MMP and Glu³⁸⁴ to Gly⁷⁹⁰ of NGFR. The other chimeramutants were also generated at the corresponding sites. All thePCR-generated fragments were confirmed by DNA sequencing.

[0231] (2) Purification and Folding of the Expressed Products

[0232] MT1EAΔ TM, MT1-PEX and MT4-PEX expressed in—E. coli were purifiedand folded as follows:

[0233] The cDNA fragments encoding Tyr¹¹² through Gly⁵³⁵ of Met plusMT1-MMP with the point mutation of Glu240 to Ala (MT1EAΔ TM), Cys³¹⁹through Gly⁵³⁵ of Met plus MT1-MMP (MT1PEX), and Cys³³⁶ through Gly⁵⁵⁰of Met plus mouse MT4-MMP were generated by PCR, and subcloned into apET3a expression vector. All the PCR-generated DNAs were confirmed byDNA sequencing.

[0234] BL21(DE3)pLys S cells were transformed with these pET3aconstructs, and protein expression was induced by 0.4 mM IPTG. Theexpressed protein products were purified and folded according to themethod of Huang et al. (Huang, W. et al., FEBS Lett. 384, 155-161,1996).

[0235] (3) Purification of proMMP-2 and TIMP-2

[0236] Recombinant human proMMP-2 and TIMP-2 were expressed in HighFiveinsect cells (Invitrogen, CA, USA), respectively, infected withrecombinant baculo viruses engineered to express human proMMP-2 andTIMP-2. Recombinant viruses were prepared using BAC-TO-BAC™ BaculovirusExpression Systems (Life Technologies, MD, USA).

[0237] Purification of proMMP-2 was conducted from the culturesupernatant by Gelatin Sepharose (Amersham Pharmacia Biotech, Inc.,Uppsala, Sweden) and gel-permeation on S-200 (Amersham PharmaciaBiotech, Inc.) according to the description in Itoh, Y. et al., Biochem.J., 308: 645-651, 1995. Purification of TIMP-2 from the culturesupernatant was conducted by Green A Dyematrex (Millipore, Co., MA, USA)and gel-permeation on S-200.

[0238] (4) Gel-Permeation Column Chromatography

[0239] MT1EAΔ TM, MT1PEX and MT4PEX were subjected to gel-permeationcolumn chromatography on Superdex 200 or Superdex 75 equilibrated with50 mM Tris-HCl buffer, pH 7.5 containing 150 mM NaCl, 10 mM CaCl₂ and0.02% NaN₃ and analyzed under the AKTA explorer 10S system (AmershamPharmacia Biotech, Inc.).

[0240] (5) Western Blotting

[0241] Cell lysate samples were subjected to separation on SDS-PAGE,followed by a transfer of the proteins in the gel to nitrocellulosemembranes (Hybond-ECL, Amersham Pharmacia Biotech, Inc.). After blockingwith a solution of 10% skim milk dissolved in TBS (20 mM Tris-HClbuffer, pH 7.5 containing 150 mM NaCl), the membrane was exposed to amonoclonal anti-FLAG M2 antibody (3 μg/ml) for detecting FLAG-taggedMT1-MMP and its mutants. In order to visualize the immunoreactive bands,the membrane was further exposed to an alkaline phosphatase-conjugatedgoat anti-mouse IgG antibody. For the purpose of detecting aphosphotyrosine residue in chimera proteins, a monoclonalanti-phosphotyrosine antibody, PY20 (1 μg/ml) was used.

[0242] (6) Gelatin Zymography

[0243] Gelatin zymography was conducted using an SDS-polyacrylamide gel(PAGE) containing gelatin (0.8 mg/ml) (Itoh, Y. et al., J. Biol. Chem.,273: 24360-24367, 1998). The samples were mixed with a SDS-PAGE loadingbuffer without a reducing agent and subjected to electrophoreticanalysis at room temperature. Enzyme activity was visualized as anegative staining area with Coomassie Brilliant Blue R-250.

[0244] (7) Cell Culture and Transfection

[0245] COS1, HT1080 and CHO-K1 cells were maintained in a HAM F-12medium or DMEM containing 10% fetal bovine serum in a humidifiedincubator at 37° C. At 16 hrs before the transfection was performed, thecells were seeded in 6-well plates at 1.5×10⁵/well.

[0246] Expression vectors for each protein were transfected usingFuGENE6™ (Roche Molecular Biochemicals) according to the manufacturer'sinstructions. At 48 hrs after the transfection was performed, the cellswere collected for use in the experiments.

[0247] (8) Construction of PEX-Deleted MT1-MMP (MT1Cat), CatalyticDomain-Deleted MT1-MMP (MT1PEX), MT1-MT4PEX, MT1-F/NGFR, MT1PEX-F/NGFR,MT1Cat-F/NGFR, and MT4PEX-F/NGFR

[0248] The cDNA of MT1-MMP was used as a template to generate DNAfragments for MT1Cat (Ile³¹⁸-Gly⁵¹⁵) and MT1PEX (Tyr¹¹²-Pro³¹²) by thePCR extension method (Ho et al., Gene, 77, 51-59 (1989)). A FLAG epitope(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-tagged MT1-MMP (MT1-F) expressionvector was constructed according to the method of Itoh et al., Biochem.J., 308: 645-651, 1999 and used as a template to generate cDNA forFLAG-tagged MT1Cat (MT1Cat-F) and MT1PEX (MT1PEX-F). The cDNA wassubcloned into pSG5 (Stratagene, CA, USA). For stable expression, thecDNA of MT1PEX-F was subcloned into pCEP4 (Invitrogen, Groningen,Netherlands) that bears EBNA-1 and hygromycin resistance gene expressioncassettes.

[0249] The DNA corresponding to Cys³¹⁹ to Cys⁵⁰⁸ in MT1-MMP and MT1PEX-Fwas replaced with Cys³³⁶ to Cys⁵²⁷ of mouse MT4-MMP to generateMT1-MT4PEX and MT4PEX-F, respectively.

[0250] A c-Myc epitope (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu)-taggedMT1-MMP (MT1-Myc) expression vector was generated by PCR in a mannersimilar to that described for MT1-F in Itoh et al., Biochem. J., 308:645-651, 1999. A c-Myc tag was inserted between Arg¹¹¹-Tyr which is thesame site for insertion of FLAG-tag in MT1-F.

[0251] Chimera mutants of the ectodomain of MT1-F, MT1Cat-F, MT1PEX-F orMT4PEX-F with the transmembrane/cytoplasmic domain of NGFR (MT1-F/NGFR,MT1Cat-F/NGFR, MT1PEX-F/NGFR, or MT4PEX-F/NGFR, respectively) were alsogenerated by PCR and subcloned into pSG5. The mutants are derived fromsequences corresponding to Met¹ through Asp⁵¹⁵ of MT1-MMP and Glu³⁸⁴through Gly¹⁹⁰ of NGFR. The other chimera mutants were also generated atthe corresponding sites. All the PCR-generated fragments were confirmedby DNA sequencing analysis.

[0252] The expression vector for the constitutively active form of Rac1(V17, Rac1DA) was kindly provided by Dr. Yoshimi Takai of OsakaUniversity.

[0253] (9) Cell Culture and Transfection

[0254] COS1 and HT1080 cells were maintained in Dulbecco's modifiedEagle's medium (DMEM; Sigma Chemical Co., Ltd., MO, USA) containing 10%fetal bovine serum and kanamycin in a humidified 37° C. incubator. At 16hrs before the transfection was performed, the cells were seeded in6-well plates at 1×10⁵/well. Each protein expression plasmid wastransfected with FuGENE6TM (Roche Molecular Biochemicals, Basel,Switzerland) according to the manufacturer's instructions.

[0255] (10) Generation of Antibodies

[0256] An anti-(MT1-MMP catalytic domain) antibody (anti-Cat) wasgenerated via immunization of rabbits with the purified MT1-MMPcatalytic domain which was expressed in E. coli. The antibody wasconfirmed to recognize MT1-MMP specifically as it did not recognizeother MT-MMPs including MT2-, MT3-, MT4-, MT5- and MT6-MMPs. Amonoclonal mouse anti-(human MT1-MMP PEX domain) antibody (anti-PEX,222-1D8) was generated via immunization of mice with a purified E.coli-expressed human MT1-MMP PEX domain. The antibody was confirmed tobe specific to MT1-MMP.

[0257] (11) Immunoprecipitation

[0258] Transfected COS1 cells were lysed in a RIPA buffer (50 mMTris-HCl, pH 7.5; 150 mM NaCl; 1% Triton-X100; 1% sodium deoxycolate;0.1% SDS; 1 mM EDTA; 1 mM PMSF; 10 μM E-64; 0.02% NaN₃) at roomtemperature for 1 hr. Cell lysates were centrifuged at 15,000 rpm inEppendorf tubes, and the supernatant was reacted with anti-FLAG M2antibody-conjugated beads (Sigma) at 4° C. for 1 hr. The beads werewashed three times with a RIPA buffer and once with Tris-buffered saline(pH 7.5) (TBS). Bound proteins were eluted by the FLAG peptide(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys: 200 μg/ml) (Sigma) in TBS, andsubjected to Western blot analysis using a monoclonal mouse anti-FLAGepitope M2 antibody (Sigma), a monoclonal mouse anti-c-Myc antibody(Ab-1) (Oncogene, MA, USA; 1 μg/ml), a rabbit anti-(human MT1Cat)antibody (1:2000), or a monoclonal mouse anti-human MT1PEX antibody(222-1D8) (0.5 μg/ml).

[0259] (12) Western blotting

[0260] Cell lysates were subjected to SDS/PAGE, followed by a transferof the proteins to nitrocellulose membranes (Hybond-ECL, AmershamPharmacia Biotech, Inc.). MT1-MMP, MT1Cat, MT1-MT4PEX and MT1-F/NGFRwere detected with a polyclonal anti-CAT antibody (1:2000), whileMT1-MMP, MT1PEX and MT1-F/NGFR with an anti-PEX antibody (222-1D8, 0.5μg/ml). FLAG-tagged MT1-MMP and its mutants were detected with ananti-FLAG M2 antibody (3 μg/ml). Chimera proteins containing aphosphotyrosine residue were detected with a monoclonal mouseanti-phosphotyrosine antibody, PY20 (ICN Biochemicals, Inc., OH, USA; 1μg/ml). Immunoreactive bands were visualized with an alkalinephosphatase-conjugated goat anti-mouse IgG or anti-rabbit IgG (Sigma).

[0261] (13) Surface Biotinylation and Immunoprecipitation

[0262] Transfected COS1 cells were washed three times with chilled PBScontaining 1 mM MgCl₂ and 0.1 mM CaCl₂. Cells were then incubated with 2mg/ml sulfo-NHS-biotin (Pierce) in the same buffer at 4° C. for 30 min.The reaction was terminated by further incubating the cells with 25 mMlysine in PBS. The cells were lysed in a RIPA buffer, and thebiotinylated proteins-were precipitated with streptavidin-Sepharosebeads (Amersham-Pharmacia). The samples were analyzed by Westernblotting using an anti-MT1Cat or anti-PEX antibody.

[0263] (14) In Situ Gelatin Degrading Assay

[0264] Two different in situ gelatin degrading assays were performed.One used DQ™ gelatin (Molecular Probes, Inc., OR, USA). DQ gelatin is afluorescence-quenched gelatin substrate, and will fluoresce uponproteolytic cleavage. Glass cover slips were coated with 5% DQ™ gelatinin a 10 mg/ml unlabeled gelatin solution, and dried. Transfected cellswere cultured on the cover slip in a humidified chamber for 16 hrs. Thecells were then fixed with 3% para-formaldehyde and the degraded areaswere visualized as those showing fluorescence.

[0265] The other assay used Alexa488-conjugated gelatin made with anAlexa488-labeling kit (Molecular Probes). The bottom surfaces of 4-wellchamber slides (Nunc) were coated with Alexa488-conjugated gelatinaccording to the method of Yana, I. and Weiss, S. J., Mol. Biol. Cell,11, 2387-2401, 2000. Transfected COS1 cells were cultured in the chamberslides for 16 hrs in a humidified culture incubator. The cells were thenfixed with 3% para-formaldehyde in PBS, and the degraded area wasanalyzed under a fluorescence microscope. The degraded area wasvisualized as a dark, non-fluorescent area. Fluorescence images wereanalyzed using confocal microscopy (BioRad).

[0266] The activities of MT1-MMPs detected by the methods describedabove are confirmed to be inhibited by the peptidyl hydroxamate MMPinhibitor, BB94. BB94 was kindly provided by Dr. Peter D. Brown ofBritish Biotech Pharmaceuticals Ltd. (Oxford, UK).

[0267] (15) Indirect Immunofluorescence Staining

[0268] Transfected COS1 cells or HT1080 cells were re-seeded ongelatin-coated cover slips and cultured for 24 hrs. The cells were thenfixed with 3% para-formaldehyde in PBS containing 75 mM lysine. Afterblocking with 5% goat serum and 3% bovine serum albumin in TBS for 1 hrat room temperature, the cells were reacted with an anti-FLAG M1antibody (5 μg/ml), PY20 (2 μg/ml), or anti-Cat antiserum (1:1,000) atroom temperature for 2 hrs. To detect the phosphotyrosine signal, thecells were permeabilized with 0.1% Triton-X100 in TBS after fixation. 1mM CaCl₂ was included throughout the procedure of washing and incubationfor staining with the anti-FLAG M1 antibody. Cy3- or Alexa488-conjugatedgoat anti-mouse IgG was used to visualize the antigen signal. Tovisualize F-actin, the cells were further incubated with Alexa488- orAlexa594-conjugated phalloidin (Molecular Probes). The signals wereanalyzed by confocal microscopy (BioRad).

[0269] (16) ProMMP-2 Binding Assay

[0270] Transfected COS1 cells were cultured in a 24-well plate(0.2×10⁴/well). Forty-eight hours after transfection, the cells werewashed three times with serum-free DMEM, and incubated with purifiedproMMP-2 (2 μg/ml) in the serum-free DMEM at 25° C. for 1 hr. The cellswere then washed three times with serum-free DMEM containing 5% dimethylsulfoxide (DMSO) to remove free proMMP-2, and lysed in 50 μl of anon-reducing SDS-loading buffer. MMP-2 bound to the cells was analyzedby gelatin zymography.

[0271] (17) Matrigel Invasion Assay

[0272] Matrigel Invasion Chambers were prepared according to themanufacturer's instructions (Becton Dickinson Labware, MA, USA) bycoating, with 10 g of Matrigel, the 8 μm-pore sized membrane of theculture inserts for 24-well plates (Falcon). A 0.5 ml aliquot oftransfected HT1080 cell suspension at 0.1×10⁵/ml was seeded on the upperchamber and incubated at 37° C. in a humidified chamber. Hepatocytegrowth factor (Toyobo; 10 ng/ml) was added to the lower chamber as achemo-attractant. After 14 hrs of incubation, cells remaining on theupper surface of the membrane were removed, and cells on the lowersurface were fixed with 3% para-formaldehyde in PBS. Green fluorescentprotein (GFP)-positive cells that had invaded to the lower, surface werecounted using fluorescence microscopy (×40).

[0273] B

[0274] The results are summarized as follows:

[0275] (1) Elucidation of Functions for the Activation of ProMMP-2 byMT1-MMP on the Cell Surface Through the Hemopexin-Like (PEX) Domain

[0276] In order to reveal the importance of the PEX domain of MT1-MMP inthe activation of proMMP-2 by an experiment, a plasmid was constructedwhich was capable of expressing PEX-deleted FLAG-tagged MT1-MMP(MT1Cat-F) (FIG. 1). In FIG. 1, each symbol indicates the following:

[0277] Pro: propeptide;

[0278] FLAG: FLAG epitope;

[0279] CD: catalytic domain;

[0280] H: hinge;

[0281] PEX: hemopexin-like domain;

[0282] TM: transmembrane domain.

[0283] The expression construct for FLAG epitope(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-tagged MT1-MMP (MT1-F) described inItoh, Y. et al., J. Biol. Chem., 274: 34260-34266, 1999 was used as atemplate to generate cDNA for MT1Cat-F (A Ile³¹⁸-Gly⁵³⁵) by the PCRextension method of Ho et al., (Ho, S. N. et al., Gene, 77: 51-59,1989). The cDNA was subcloned into pSG5 (Stratagene, CA, USA). ThePCR-generated fragments were confirmed by DNA sequencing. COS1 cellswere transfected with the expression vector for MT1-MMP mutant; MT1-F orMT1Cat-F, or a vector alone (Mock). Expression vectors for each proteinwere transfected using FuGENE6™ (Roche Molecular Biochemicals) accordingto the manufacturer's instructions. COS1 cells were seeded in 6-wellplates at 1.5×10⁵/well at 16 hrs before the transfection was performed.The COS1 cells were maintained in a HAM F-12 medium or DMEM containing10% fetal bovine serum in a humidified incubator at 37° C. At 48 hrsafter the transfection, the cells were collected for use in theexperiments. The cells were incubated in the presence of purifiedproMMP-2 in a serum-free medium (0.25 μg/ml) at 37° C. for 18 hrs. Celllysates were tested for the expression of the mutants, while culturesupernatants were tested for the activation of proMMP-2.

[0284] The COS1 cells transfected with FLAG-tagged full length MT1-MMPactivated exogenous proMMP-2 which was added to the culture supernatantwhile the cells transfected with MT1Cat-F did not activate at all (FIG.2). The upper panel in FIG. 2 shows Western blotting analysis using amonoclonal anti-FLAG M2 antibody for cell lysates obtained from thetransfected cells. The lower panel in FIG. 2 shows gelatin zymography ofthe culture medium.

[0285] MT1Cat-F is expressed on the cell surface, and has similargelatin degrading activity as compared to MT1-F (see, FIG. 13, lowerpanel). Therefore, it is obvious that differences in the activation ofproMMP-2 results from either its appearance manner on the cell surfaceor its proteolytic degradation activity. PEX is thought to decidepresumably the arrangement of MT1-MMP on the cell surface throughcomplex formation.

[0286] (2) To Test Whether the Function of MT1-MMP Ectodomain is Presentor Not

[0287] In order to test if the ectodomain of MT1-MMP is involved in thecomplex formation, a catalytically-inactive soluble mutant (MT1EAΔ TM)wherein a propeptide was deleted was expressed in E. coli (FIG. 3). ThecDNA fragments encoding Tyr¹¹² through Gly⁵³⁵ of Met plus MT1-MMP withthe point mutation of Glu240 to Ala (MT1EAΔ TM) was prepared by PCR, andsubcloned into a pET3a expression vector. The resulting PCR-generatedDNA was confirmed by DNA sequencing. E. coli BL21(DE3)pLys S cells weretransformed with the pET3a construct thus prepared, and proteinexpression was induced by 0.4 mM IPTG. The expressed protein product waspurified and folded according to the method of Huang et al. (Huang, W.et al., FEBS Lett. 384, 155-161, 1996). This purified protein appearedto be more than 98% pure, as shown in FIG. 4.

[0288] Although the catalytically active site was mutated so as to avoidauto-degradation, this mutation should not create an overall structuralalteration, as has been shown in other MMPs (Atkinson, S. J. et al., J.Biol. Chem., 270: 30479-30485, 1995). Furthermore, MT1EA A TM wassubjected to Superdex 200 gel filtration column chromatography at threedifferent concentrations. The inset in FIG. 4 shows the SDS-PAGEanalyses of MT1EA Δ TM under reducing and non-reducing conditions. Bandswere visualized with Coomassie Blue R-250.

[0289] MT1EAΔ TM demonstrated a different mobility under reducing andnon-reducing SDS-PAGE, suggesting that the S—S bond was correctlyformed. In addition, it was confirmed that MT1EAΔ TM had an ability toform a complex with TIMP-2, suggesting that this catalytic domain wascorrectly folded. For the purification step, it was noted that MT1EAΔ TMdid not elute from the gel permeation column where expected for its sizeof 53 kDa, but rather at a volume suggesting a larger molecular mass(600 kDa or greater, and 100 kDa).

[0290] Accordingly, it was suggested that MT1-MMP formed a homophiliccomplex. This complex formation (in particular, the formation of acomplex having a high molecular mass) is dependent on the concentrationof MT1EAΔ TM (FIG. 4). At a concentration of 0.33 μM, a monomer peak at50 kDa and a peak above 600 kDa were observed. When the sample was addedat 0.66 μM or 1.4 μM, the height of a dimer peak around 130 kDaincreased, while the height of a peak corresponding to a highermolecular mass further increased (FIG. 4). The appearance of a dimer, atetramer, and additional polymers was also revealed for MT1EAΔ TMtreated with a chemical crosslinking agent DSG (FIG. 5). Crosslinking ofMT1EAΔ TM was conducted as follows:

[0291] Purified MT1EAΔ TM derived from E. coli was treated with 0.2 mMDSG in 50 mM HEPES containing 150 mM NaCl, 10 mM CaCl₂, 0.02% NaN₃ at 0°C. The reaction was terminated by adding a Tris-HCl buffer, pH 7.5 untilits final concentration reached 10 mM.

[0292] (3) Test for a Domain Involved in the Formation of a Complex

[0293] To determine whether either of the catalytic domain and PEX wasinvolved in the complex formation, MT1EAΔ TM was treated with trypsin.Since trypsin is found to cleave the middle of the MT1EAΔ TM hingeregion that contains several Arg and Lys, it is capable of separatingthe catalytic domain and PEX. The sample was then analyzed on gelpermeation column chromatography with Superdex 75. Thus, MT1EAΔ TM (50μg/ml) was treated with trypsin (0.2 μg/ml) at 37° C. for 1 hr to cleavebetween the catalytic domain and PEX. Trypsin was deactivated by adding2 mM PMSF to the mixture. Next, the sample was applied to a Superdex 75gel filtration column. As shown in FIG. 6, the catalytic domain waseluted at a volume corresponding to around 17 kDa, but PEX was eluted ata volume corresponding to around 40 kDa. Since the catalytic domain andPEX migrate in SDS-PAGE under non-reducing conditions to 21 kDa and 24kDa, respectively (FIG. 6), it is suggested that PEX but not thecatalytic domain forms a homophilic complex in solution.

[0294] Moreover, to test if the complex formation of PEX is specific toMT1-MMP, Cys³¹⁹ through Gly⁵³⁵ of Met plus MT1-MMP (MT1PEX) and the PEXof MT4-MMP were expressed in E. coli. The cDNA fragments encodingMT1PEX, and Cys³³⁶ through Gly⁵⁵⁰ of Met plus mouse MT4-MMP (GenBank™accession number AB021224) were prepared by PCR, and subcloned into apET3a expression vector. The PCR-generated DNA was confirmed by DNAsequencing. Thereafter, E. coli BL21(DE3)pLys S cells were transformedwith the pET3a constructs, and protein expression was induced by 0.4 mMIPTG. The expressed protein products were purified and folded accordingto the method of Huang et al. (Huang, W. et al., FEBS Lett. 384,155-161, 1996).

[0295] As shown in FIG. 7, the apparent molecular mass of the PEX ofMT4-MMP (MT4-PEX) is almost identical to that of the PEX of MT1-MMP(MT1-PEX) on SDS-PAGE under reducing and non-reducing conditions (FIG.6). However, on gel-permeation chromatography, the PEX of MT4-MMP(MT4-PEX) eluted at approximately 24 kDa whereas the PEX of MT1-MMP(MT1-PEX) eluted at approximately 40 kDa, suggesting that dimer complexformation was specific for MT1-PEX. These results indicate that theectodomain of MT1-MMP forms a homophilic complex through the interactionof its PEX at least in a solution.

[0296] In other words, the ectodomain of MT1-MMP forms a homophiliccomplex through the interaction of PEX thereof in vitro.

[0297] (4) Test for a Site Where a Homophilic Complex is Formed

[0298] Next, it is tested whether MT1-MMP expressed in animal cells alsoforms a homophilic complex or not. For the test, the trk protooncogene(nerve growth factor receptor, NGFR) is utilized in order to monitordimer formation on the cell surface.

[0299] The principle of assay is as follows:

[0300] NGFR forms a dimer through binding to its ligand. Subsequently,tyrosine residues in the cytoplasmic domain are trans-phosphorylated byits tyrosine kinase (Jing, S. et al., Neuron, 9: 1067-1079, 1992;Lemmon, M. A. et al., Trends Biochem. Sci., 19: 459-463, 1994). Thus, itwas demonstrated that the fusion protein between (a) the cytoplasmicdomain of NGFR and (b) BCR of the oncoprotein BCR-ABL inducedconstitutive phosphorylation of NGFR's tyrosine residues when BCR formeda tetramer (Maru, Y. et al., Oncogene, 16: 2585-2595, 1998).

[0301] By utilizing the above principle, a chimeric protein is firstconstructed consisting of the ectodomain of MT1-MMP and thetransmembrane (TM) and cytoplasmic (CP) domains of NGFR (FIG. 8) inorder to test for direct complex formation. When the ectodomain of thischimeric protein forms a homophilic complex in a similar manner,tyrosine residues in the cytoplasmic domain will be phosphorylated.

[0302] Accordingly, to test for domain specific interactions of MT1-MMP,the following chimeras were constructed (FIG. 8):

[0303] MT1-F/NGFR; MT1PEX-F/NGFR; MT1Cat-F/NGFR; MT4PEX-F/NGFR

[0304] In FIG. 8, each of the symbols other than those described aboveis as follows:

[0305] NGFR-TM/CP, transmembrane and cytoplasmic domains derived fromNGFR;

[0306] TK, tyrosine kinase domain;

[0307] Y, tyrosine residues phosphorylated.

[0308] A FLAG-tagged MT1-MMP (MT1-F) expression construct as used hereinwas as described in Itoh, Y. et al., J. Biol. Chem. 274: 34260-34266,1999. In addition, MT1-F was used as a template to generate cDNAs forMT1Cat-F (Δ Ile³¹⁸-Gly⁵³⁵) and MT1PEX-F (ΔTyr¹¹²-Pro³¹²) by the PCRextension method of Ho et al. (Ho, S. N. et al., Gene, 77: 51-59, 1989).The cDNA was subcloned into pSG5 (Stratagene, CA, USA). Chimera mutantsof the ectodomain of MT1-F, MT1Cat-F, MT1PEX-F or MT4PEX-F, with thetransmembrane (TM)/cytoplasmic (CP) domain of NGFR were also generatedby the PCR method in a similar manner, and subcloned into pSG5. TheMT1-MMP-derived sequence corresponded to Met¹ through Asp⁵¹⁵ of MT1-MMP,while the NGFR-derived sequence corresponded to Glu³⁸⁴ through Gly⁷⁹⁰ ofNGFR (GenBank™ accession number M23102). The other chimera mutants werealso composed from these corresponding sequences. All the PCR-generatedfragments were confirmed by DNA sequencing.

[0309] Expression vectors for the protein were transfected into COS1cells, using FuGENE6™ (Roche Molecular Biochemicals) according to themanufacturer's instructions. COS1 cells were seeded in 6-well plates at1.5×10⁵/well at 16 hrs before the transfection was performed. The COS1cells were maintained in a HAM F-12 medium or DMEM containing 10% fetalbovine serum in a humidified incubator at 37° C. At 48 hrs after thetransfection, the cells were collected for use in the experiments.

[0310] As shown in FIG. 9, these proteins expressed via the transfectionof these constructs were recognized by an anti-FLAG M2 antibody (upperpanel, Anti-FLAG). When identical samples were analyzed with ananti-phosphotyrosine antibody (Anti-PY), tyrosine residues of MT1-F/NGFRwere phosphorylated (lower panel, Anti-PY, lane 1), suggesting thatMT1-MMP expressed in animal cells also formed at least a dimer.Additionally, MT1PEX-F/NGFR was phosphorylated (lane 2) as well.

[0311] In contrast, sufficient phosphorylation was not observed forcells transfected with MT1Cat-F/NGFR or MT4PEX-F/NGFR (lanes 3 and 4).

[0312] These results strongly support the complex formation through PEXas found in vitro. In other words, it is clear that MT1-MMP forms ahomophilic complex on the cell surface.

[0313] (5) Inhibition of Dimer Formation by Co-expression With MT1PEX-F

[0314] It is investigated whether co-expression of MT1PEX-F and MT1-Fcompetes in the dimer formation of MT1-F/NGFR or not. For the test, theexpression plasmids for MT1-F/NGFR and MT1PEX-F were co-transfected intoCOS1 cells.

[0315] As shown in FIG. 10, phosphorylation of MT1-F/NGFR (lane 1) wasinhibited depending on the expression level of MT1PEX-F (lanes 2-5).

[0316] (6) Elucidation of the Significance of MT1-MMP Complex Formationon the Cell Surface

[0317] To investigate roles of dimer formation upon the activation ofproMMP-2, MT1-F was introduced into COS1 cells together with MT1PEX-F.The co-introduced cells were reacted with exogenously added proMMP-2.Thus, COS1 cells were co-transfected with the MT1-F and MT1PEX-F atindicated DNA amount ratios. Next, purified proMMP-2 (0.25 μg/ml) in aserum-free medium was reacted with the resulting cells at 37° C. for 18hrs. MMP-2 in the culture supernatant was analyzed by gelatinzymography, and the cell lysates were subjected to Western blottinganalysis using a monoclonal anti-FLAG M2 antibody.

[0318] As shown in FIGS. 11 and 28, the expression of MT1PEX-F inhibitedthe activation of proMMP-2 by MT1-F, in dependence upon the expressionlevel. This observation suggests that the dimer formation is necessaryfor the activation of proMMP-2 on the cell surface, and that MT1PEX-Fcan inhibit the function of MT1-MMP in a dominant negative manner.

[0319] Next, to determine if MT1PEX-F affects the catalytic activity ofMT1-MMP on the cell surface or not, tests for gelatin degrading activityand binding capacity to TIMP-2 were conducted using cells transfected inthe same incubator. As shown in FIGS. 12 through 14, CHO-K1 cells weretransfected with an expression vector for MT1-F, MT1-F/MT1PEX-F orMT1Cat-F, or a vector alone (Mock). The cells were then treated withtrypsin, and separately seeded to: DQ™ gelatin-coated cover slips fortesting the proteolytic activity (FIG. 13); and 24-well plates forTIMP-2 binding assay (FIG. 12) and for the activation of proMMP-2 (FIG.14). Transfected cells on the DQ™ gelatin-coated cover slip as shown inFIG. 13 were incubated in a medium at 37° C. for 18 hrs, and thegenerated fluorescence was analyzed using confocal microscopy.Transfected cells on the 24-well plate as shown in FIG. 12 wereincubated with 25 nM ¹²⁵I-labeled TIMP-2 in the presence or absence ofBB-94 (50 μM) at 0° C. for 1 hr. MT1-MMP specific binding was calculatedby subtracting the datum obtained in the presence of BB-94 (50 μM) fromeach datum derived from the corresponding experiments. Identicallytransfected cells on the 24-well plate as shown in FIG. 14 weresubjected to the reaction with purified proMMP-2 in a serum-free mediumat 37° C. for 18 hrs. The cells and culture supernatants wererespectively subjected to Western blotting analysis using a monoclonalanti-FLAG M2 antibody, and gelatin zymography analysis. All results werederived from the identical transfection experiments. DQ™ gelatin is afluorescence-quenched gelatin, which fluoresces upon proteolyticcleavage. This characteristic was utilized for the test.

[0320] As shown in FIG. 13, all of the cells transfected with MT1-F,MT1-F/MT1PEX-F, or MT1Cat-F exhibited the gelatin degrading activity,and fluoresced, suggesting that all of them retain proteolytic activity.Although the co-transfection of MT1PEX-F led to a 37% decrease in TIMP-2binding on the cell surface as compared with MT1-F alone (FIG. 12), itcould not be explained why these cells completely inhibited theactivation of proMMP-2 (FIG. 14). MT1Cat-F-expressing cells exhibited anequivalent TIMP-2 binding capacity as compared to the cells bearingMT1-F (FIG. 12).

[0321] These results indicate that the dimer formation through the PEXof MT1-MMP is of importance in the activation of proMMP-2 on the cellsurface.

[0322] To test if MT1PEX-F also functions in biological systems,Matrigel invasion by MT1-MMP-expressing HT1080 cells was examined.

[0323] Cells were transiently transfected with a vector for MT1PEX-F,together with an expression vector for green fluorescent protein (GFP),and GFP-positive invading cells were counted under fluorescencemicroscopy. HT1080 cells were co-transfected with expression vectors forMT1PEX-F and GFP, or with an expression vector for GFP alone. The cellswere assayed for Matrigel invasion as described below. The modifiedBoiden-chamber method was carried out.

[0324] Matrigel Invasion Chambers were coated with 10 g of Matrigel perchamber according to the manufacturer's instructions (Becton Dickinson),and were placed on the 8 μm pore-sized membrane of culture inserts(FALCON). A 0.5 ml aliquot of HT1080 cell suspension at 1×10⁵/ml wasseeded on the upper chamber, and incubated at 37° C. in a humidifiedchamber for 14 hrs. HGF (5 ng/ml) was added to the lower chamber as achemo-attractant.

[0325] TIMP-2 at 0.5 μg/ml was added to the upper and lower chambers.GFP-positive cells on the surface of the lower chamber were countedusing fluorescence microscopy. The number of GFP-positive cells amongmock-transfected cells was 1.31×10⁴/1×10⁵ cells. The number ofGFP-positive cells among MT1PEX-F-transfected cells was 1.12×10⁴/1×10⁵cells.

[0326] As shown in FIG. 15, exogenously added TIMP-2 inhibited by 50%the invasion by mock-transfected cells. In contrast, by transfectionwith the expression plasmid for MT1PEX-F, the invasion was suppressed by70%. The addition of TIMP-2 to the MT1PEX-F-expressing cells produced nofurther reduction in the invasion. These results indicate that MT1PEX-Fcan inhibit not only the transiently expressed MT1-MMP activity but alsothe endogenous MT1-MMP activity. Accordingly, the expression of PEX inHT1080 cells significantly reduced Matrigel invasion activity.

[0327] In other words, these findings indicate the action mechanism ofMT1-MMP to activate proMMP-2 on the cell surface, and also reveal thatthis mechanism finally promotes cell invasiveness in tissues. Thus, itis suggested that dimer formation of MT1-MMP is essential for theactivation of proMMP-2, leading to the promotion of tumor cell invasion.

[0328] (7) Homophilic Complex Formation of MT1-MMP

[0329] The activation of proMMP-2 by MT1-MMP on the cell surfacerequires the ternary complex formation of proMMP-2, TIMP-2 and MT1-MMP,and also the presence of TIMP-2-free MT1-MMP near the ternary complex.If TIMP-2-free MT1-MMP and the ternary complex are far apart, theactivation should not occur. We thus hypothesized that MT1-MMP wouldform a homophilic complex on the cell surface to arrange the effectiveactivation complex for MMP-2. To test this possibility, FLAG-taggedMT1-MMP (MT1-F) and c-Myc-tagged MT1-MMP (MT1-Myc) (FIG. 16) wereco-expressed in COS1 cells, and MT1-F was immunoprecipitated withanti-FLAG M2 antibody-conjugated beads. As shown in FIG. 17, MT1-Myc wasco-immunoprecipitated with MT1-F when co-expressed with MT1-F (lane 6,Anti-FLAG and Anti-Myc), whereas MT1-Myc alone did not lead toimmunoprecipitation (lane 7, Anti-Myc). This indicates that the MT1-MMPmolecules having different tags formed a complex with each other. Next,to test the domain requirement for the complex formation, MT1-F wasco-expressed either (i) with MT1-MMP lacking the PEX domain (MT1Cat) or(ii) with MT1-MMP lacking the catalytic domain (MT1PEX) (FIG. 16), andsubjected to immunoprecipitation with anti-FLAG beads. The samples werethen subjected to Western blotting using anti-FLAG M2, anti-MT1PEX, andanti-MT1Cat to examine the species co-immunoprecipitated with MT1-F. Asshown in FIG. 18, MT1PEX but not MT1Cat was co-immunoprecipitated withMT1-F (Anti-PEX, lane 9 and Anti-Cat, lane 8, respectively). Thisindicates that MT1-MMP forms a homophilic complex through the PEXdomain.

[0330] (8) Necessity of PEX in the Activation of ProMMP-2 by MT1-MMP onthe Cell Surface

[0331] To confirm the importance of the MT1-MMP PEX domain in theactivation of proMMP-2, the MT1-MMP PEX domain was exchanged with theone derived from MT4-MMP (FIG. 19, MT1-MT4PEX). As shown in FIG. 20,MT1-MT4PEX-expressing COS1 cells did not activate proMMP-2 (upper panel)although the expression level was similar to that of the wild typeenzyme (second panel, Anti-Cat). MT1-MT4PEX is expressed on the cellsurface, as the molecule was sensitive to surface biotinylation (lowerpanel, Surface Biotinylation). MT1-MT4PEX can form a ternary complex ofMT1-MT4PEX/TIMP-2/proMMP-2 on the cell surface as the proMMP-2 bound toMT1-MT4PEX-expressing cells (FIG. 21, lanes 2 and 3). To examine ifMT1-MT4PEX is proteolytically active on the cell surface, thetransfected cells were cultured on DQ™ gelatin substrate. DQ™ gelatin isa fluorescence-quenched substrate that fluoresces upon proteolyticcleavage. As shown in FIG. 22, MT1-MMP-as well as MT1-MT4PEX-expressingcells showed proteolytic activity, whereas mock-transfected cells (Mock)did not. This activity was also completely inhibited by BB94. It hasbeen confirmed that the PEX domain-deleted MT1-MMP (MT1Cat) cannotactivate proMMP-2, although it retains its proteolytic activity as wellas its ability to form the ternary complex of MT1Cat/TIMP-2/proMMP-2 onthe cell surface, as does MT1-MT4PEX. Taken together, these data showthat the dimer formation of MT1-MMP through the PEX domain on the cellsurface is required to activate proMMP-2.

[0332] (9) MT1-MMP Forms a Homophilic Complex on the Cell Surface

[0333] To monitor the dimer formation of the enzyme in the cells,another approach to detect the molecular interactions was executed.Growth factor receptors, including nerve growth factor receptor (NGFR),form a dimer or oligomer complex through the ectodomain to bind to theirspecific ligands. Subsequently, the cytoplasmic domains containingtyrosine kinase (TK) are brought into close proximity. It is thuspossible to determine the specific homodimer interactions of a proteinby making a chimeric protein with a receptor molecule. For example, thefusion protein between the cytoplasmic domain of NGFR and BCR of theoncoprotein BCR-ABL which forms a tetramer exhibited constitutivephosphorylation of tyrosine residues. Accordingly, a chimeric proteinconsisting of the ectodomain of MT1-MMP and the transmembrane andcytoplasmic domains of NGFR was constructed (FIG. 8, MT1-F/NGFR).

[0334] To test for domain-specific interactions in this system, thefollowing chimeras were constructed:

[0335] MT1PEX-F/NGFR; MT1Cat-F/NGFR; and MT4PEX-F/NGFR (FIG. 8).

[0336] These constructs were expressed in COS1 cells at levels similarto MT1-F/NGFR expression as detected by an anti-FLAG M2 antibody (FIG.9, upper panel, Anti-FLAG, lanes 2-4). When tyrosine phosphorylation inthe identical samples was detected by PY, only MT1PEX-F/NGFR was shownto be strongly phosphorylated (lower panel, Anti-PY, lane 2).Phosphorylation levels of MT1Cat-F/NGFR and MT4PEX-F/NGFR were extremelylow (lanes 3 and 4). These results indicate that a homophilic complexwas formed through PEX on the cell surface, supporting the foregoingdata.

[0337] Next, the expression plasmids for MT1-F/NGFR and FLAG-taggedMT1PEX (MT1PEX-F), MT1Cat (MT1Cat-F), or MT1-F were co-transfected toinvestigate if the dimer formation of the chimera could be disrupted ornot. As shown in FIG. 23, co-expression of MT1PEX-F and MT1-Feffectively competed in the dimer formation of MT1-F/NGFR (lanes 2 and4).

[0338] In contrast, the expression of MT1Cat-F did not decrease theintensity of the band (lane 3). Furthermore, 90% or more inhibition ofthe phosphorylation was observed by increasing levels of MT1PEX-Fexpression (FIG. 10, lanes 1-5). This further suggests that MT1-MMPmolecules interact through the PEX domains.

[0339] (10) Dimer Formation of MT1-MMP Increases ProMMP-2 Activation

[0340] Using the chimera construct, it was investigated whether thereare any regulatory factors that might regulate the dimer formation ofMT1-MMP on the cell surface. Since it has been reported that MT1-MMP islocalized at the lamellipodia structure in walking osteoclasts, it hasbeen examined for the efficacy of a constitutively active form of thesmall GTPase Racl (V12Racl, Rac1DA) on the dimer formation as aGTP-bound form of Racl stimulates the generation of lamellipodia incells. MT1-F/NGFR was co-expressed with Rac1DA in COS1 cells and thephosphotyrosine signal was monitored. As shown in FIG. 24,co-transfection of Rac1DA and MT1-F/NGFR resulted in an enhancedphosphotyrosine signal as compared to MT1-F/NGFR alone (Anti-PY, lanes 1and 3). When MT1PEX-F was co-expressed, the signal decreased by 43%,suggesting that the increased phosphotyrosine signal was the result ofenhanced dimer formation, but was not likely to be due to other tyrosinekinases which might be present downstream of the Rac1 signal, asMT1PEX-F inhibits dimer formation (Anti-PY, lanes 2 and 4, see alsoFIGS. 9, 10 and 23). Immunostaining of MT1-F/NGFR-expressing cells forphosphotyrosine revealed that distinct signals were detected in thecells, especially where the F-actin was concentrated (FIG. 25,MT1-F/NGFR). Expression of Rac1DA generated ruffled membranes (FIG. 25,MT1-F/NGFR•Rac1DA and Rac1DA). Surprisingly, phosphotyrosine signalslocalized extensively at the edge of the ruffled membrane structure(MT1-F/NGFR•Rac1DA). These signals were confirmed to be generated byMT1-F/NGFR as they were co-localized with the staining pattern with theanti-FLAG M1 antibody, and no such signals were detected in Rac1DA- orcontrol-transfected cells (Rac1DA and Mock). These results suggest thataccelerated dimer formation occurs at the ruffled membrane edge. Then,the effect of Rac1DA expression on the proMMP-2 activation by MT1-MMPwas investigated. As shown in FIG. 26, co-expression of MT1-MMP andRac1DA clearly enhanced the processing of proMMP-2 to provide its activeform. Immunolocalization of MT1-F in the transfected cells shows thatMT1-F is distributed on the cell surface and is relatively concentratedat the lamellipodium structure where F-actin is concentrated (FIG. 27,Anti-FLAG, MT1-F).

[0341] In contrast, co-expression of Rac1DA with MT1-F reinforced thelocalization of MT1-F at the ruffled membrane edge (Anti-FLAG,MT1-F/Rac1DA). These data suggest that the expression of RaclDAincreases MT1-MMP dimer formation, and the activation of proMMP-2 bygenerating membrane ruffles that result in an increased concentration ofMT1-MMP at the site of the plasma membrane.

[0342] (11) Complex Formation of MT1-MMP on the Cell Surface isImportant for MT1-MMP's Biological Activity

[0343] Next, the efficacy of MT1PEX-F on the activation of proMMP-2 wasexamined, since MT1PEX-F effectively inhibited the dimer formation ofMT1-F/NGFR. COS1 cells were transfected with the plasmids for MT1-MMPand MT1PEX-F in different ratios.

[0344] As shown in FIG. 29, proMMP-2 binding to the cell surface wasalso not affected by MT1PEX-F expression. Co-expression of MT1PEX-F didnot decrease the amount of proMMP-2 bound to the cell surface, and noactive form was detected. MT1PEX-F also did not affect the proteolyticactivity of MT1-MMP on the cell surface as shown in FIG. 30, showing asimilar gelatin degradation pattern and area. Therefore, MT1PEX-Finhibits the dimer formation of MT1-MMP and acts as a dominant negativeform of MT1-MMP in the activation of proMMP-2 on the cell surface.

[0345] HT1080 cells are known to express MT1-MMP and MMP-2 endogenously.Thus, the efficacy of MT1PEX-F expression on the activation ofendogenous proMMF-2 in HT1080 cells was next examined. The cells weretransfected with a plasmid for MT1PEX-F or an empty vector bearing thehygromycin-resistant gene. The culture medium from thehygromycin-resistant cells was analyzed for produced MMP-2 byzymography. As shown in FIG. 31, control cells activate endogenous MMP-2in a manner sensitive to BB94 (upper panel, lanes 1 and 2) while theMT1PEX-F-expressing cells did not effectively activate proMMP-2 (upperpanel, lane 3). Western blotting analysis of the cell lysates indicatedthat HT1080 cells spontaneously produced MT1-MMP, and MT1-MMP waspartially processed to a 43 kDa fragment as reported previously.MT1PEX-F did not affect the expression and processing of endogenousMT1-MMP. Co-expression of endogenous MT1-MMP and MT1PEX-F clearlyindicated the co-localization of both molecules at the edge of the cells(FIG. 32).

[0346] In accordance with the present invention, it was reveled thatMT1-MMP forms a homodimer through MT1-PEX, and that the complexformation is essential for the activation of progelatinase A (proMMP-2)on the cell surface. Similarly to the requirement of TIMP-2 for theactivation of proMMP-2, it is clear that cell surface MT1-MMP requiresthe dimer formation as demonstrated in the present invention. TIMP-2which is bound to MT1-MMP is necessary to concentrate proMMP-2 on thecell surface where the activation occurs. For activating proMMP-2 boundto TIMP-2/MT1-MMP complex, MT1-MMP which is not inhibited by TIMP-2should be located close thereto.

[0347] As demonstrated in the present invention, MT1-MMP without PEX(MT1Cat-F) can no longer activate proMMP-2. Since Mt1Cat-F-expressingcells showed gelatinase activity and TIMP-2 binding capacity on theircell surface, it is clear that the said MT1Cat-F defect of not beingable to activate proMMP-2 is not due to the lack of proteolytic activityand of binding capacity to TIMP-2. Therefore, the activation is believedto result from random distribution of MT1Cat-F on the cell surface.

[0348] Accordingly, it is conceivable that dimer formation of MT1-MMPthrough PEX on the cell surface allows a closer molecular arrangementbetween two enzyme molecules and that one of the MT1-MMP molecules inthe dimer complex binds to TIMP-2 to act as a proMMP-2 receptor whilethe other MT1-MMP molecule cleaves the propeptide of proMMP-2 bound tothe receptor.

[0349] MT1PEX-F creates a certain distance between the MT1-MMP/TIMP-2complex and the catalytic MT1-MMP so that the reaction of proMMP-2 withthe enzyme for its activation is prevented.

[0350] It was previously reported that a catalytic domain-deleted formof MT1-MMP was generated in HT1080 cells (Stanton, H. et al., J. CellSci., 111: 2789-2798, 1998; Lehti, K. et al., Biochem. J., 334: 345-353,1998). This product is considered to be the result of a specificcleavage at the Ala²⁵⁵-Lle bond located at the end of the MT1-MMPcatalytic domain by the action of either MT1-MMP itself or MMP-2activated by MT1-MMP.

[0351] Since this deleted form retains PEX, it may have a similar effectto MT1PEX-F. Thus, this processing not only removes the activity ofMT1-MMP, but may also inhibit adjacent MT1-MMP activity.

[0352] In accordance with the present invention, it has been revealedthat PEX plays an important role in MT1-MMP dimer formation and furtherfunctions in facilitating effective activation of proMMP-2 on the cellsurface. Therefore, it is conceivable that PEX is a uniquely preserveddomain in matrixins and a general device involved in interactions withmolecules that determine the fate of the enzyme in biological systems.

[0353] Accordingly, roles of proMMP-2 activation or MT1-MMP dimerformation in specific biological systems can be tested using MT1PEX-F.In addition, since Matrigel invasion activity owned by HT1080 cellscapable of producing MT1-MMP and MMP-2 (Strongin, A. Y. et al., J. Biol.Chem., 270: 5331-5338, 1995) is effectively inhibited by MT1PEX-F, itcan be utilized in developing and investigating means with intent tosuppress and/or inhibit the invasion and/or metastasis of cancer cells.Furthermore, it has been shown that MT1PEX-F blocks the MT1-MMPfunctions without affecting MT1-MMP proteolytic activity. MT1-F/tfkhaving NGFR transmembrane/cytoplasmic domains forms a dimer complex, andthis complex formation is inhibited by MT1PEX-F, suggesting that asequence property of the transmembrane/cytoplasmic domains is notimportant for dimer formation. Moreover, GPI-anchored MT1PEX-F inhibitsthe activation of proMMP-2. Thus, the inhibitory efficacy of MT1PEX-F istruly a result of PEX interaction.

INDUSTRIAL APPLICABILITY

[0354] In accordance with the present invention, the activationmechanisms of proMMP-2 by MT1-MMP existing on the cell surface have beendisclosed, thereby enabling the investigation and development ofsubstances active in affecting the activation of proMMP-2. Moreover, theactivation of proMMP-2 by MT1-MMP existing on the cell surface issuspected to participating in a variety of physiological or biologicalphenomena, for example, disorders, abnormal conditions and/or diseasesin living organisms, including invasion or metastasis of cancer cells.Therefore, it paves the way to serve in prophylaxis and/or therapy ofthese phenomena.

[0355] While the present invention has been described specifically indetail with reference to certain embodiments and examples thereof, itwould be apparent that it is possible to practice it in other forms. Inlight of the disclosure, it will be understood that variousmodifications and variations are within the spirit and scope of theappended claims.

1 20 1 8 PRT Artificial Sequence Description of Artificial Sequence FLAGepitope 1 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 2 590 PRT ArtificialSequence Description of Artificial Sequence MT1-F 2 Met Ser Pro Ala ProArg Pro Ser Arg Cys Leu Leu Leu Pro Leu Leu 1 5 10 15 Thr Leu Gly ThrAla Leu Ala Ser Leu Gly Ser Ala Gln Ser Ser Ser 20 25 30 Phe Ser Pro GluAla Trp Leu Gln Gln Tyr Gly Tyr Leu Pro Pro Gly 35 40 45 Asp Leu Arg ThrHis Thr Gln Arg Ser Pro Gln Ser Leu Ser Ala Ala 50 55 60 Ile Ala Ala MetGln Lys Phe Tyr Gly Leu Gln Val Thr Gly Lys Ala 65 70 75 80 Asp Ala AspThr Met Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 85 90 95 Asp Lys PheGly Ala Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Asp 100 105 110 Tyr LysAsp Asp Asp Asp Lys Tyr Ala Ile Gln Gly Leu Lys Trp Gln 115 120 125 HisAsn Glu Ile Thr Phe Cys Ile Gln Asn Tyr Thr Pro Lys Val Gly 130 135 140Glu Tyr Ala Thr Tyr Glu Ala Ile Arg Lys Ala Phe Arg Val Trp Glu 145 150155 160 Ser Ala Thr Pro Leu Arg Phe Arg Glu Val Pro Tyr Ala Tyr Ile Arg165 170 175 Glu Gly His Glu Lys Gln Ala Asp Ile Met Ile Phe Phe Ala GluGly 180 185 190 Phe His Gly Asp Ser Thr Pro Phe Asp Gly Glu Gly Gly PheLeu Ala 195 200 205 His Ala Tyr Phe Pro Gly Pro Asn Ile Gly Gly Asp ThrHis Phe Asp 210 215 220 Ser Ala Glu Pro Trp Thr Val Arg Asn Glu Asp LeuAsn Gly Asn Asp 225 230 235 240 Ile Phe Leu Val Ala Val His Glu Leu GlyHis Ala Leu Gly Leu Glu 245 250 255 His Ser Ser Asp Pro Ser Ala Ile MetAla Pro Phe Tyr Gln Trp Met 260 265 270 Asp Thr Glu Asn Phe Val Leu ProAsp Asp Asp Arg Arg Gly Ile Gln 275 280 285 Gln Leu Tyr Gly Gly Glu SerGly Phe Pro Thr Lys Met Pro Pro Gln 290 295 300 Pro Arg Thr Thr Ser ArgPro Ser Val Pro Asp Lys Pro Lys Asn Pro 305 310 315 320 Thr Tyr Gly ProAsn Ile Cys Asp Gly Asn Phe Asp Thr Val Ala Met 325 330 335 Leu Arg GlyGlu Met Phe Val Phe Lys Lys Arg Trp Phe Trp Arg Val 340 345 350 Arg AsnAsn Gln Val Met Asp Gly Tyr Pro Met Pro Ile Gly Gln Phe 355 360 365 TrpArg Gly Leu Pro Ala Ser Ile Asn Thr Ala Tyr Glu Arg Lys Asp 370 375 380Gly Lys Phe Val Phe Phe Lys Gly Asp Lys His Trp Val Phe Asp Glu 385 390395 400 Ala Ser Leu Glu Pro Gly Tyr Pro Lys His Ile Lys Glu Leu Gly Arg405 410 415 Gly Leu Pro Thr Asp Lys Ile Asp Ala Ala Leu Phe Trp Met ProAsn 420 425 430 Gly Lys Thr Tyr Phe Phe Arg Gly Asn Lys Tyr Tyr Arg PheAsn Glu 435 440 445 Glu Leu Arg Ala Val Asp Ser Glu Tyr Pro Lys Asn IleLys Val Trp 450 455 460 Glu Gly Ile Pro Glu Ser Pro Arg Gly Ser Phe MetGly Ser Asp Glu 465 470 475 480 Val Phe Thr Tyr Phe Tyr Lys Gly Asn LysTyr Trp Lys Phe Asn Asn 485 490 495 Gln Lys Leu Lys Val Glu Pro Gly TyrPro Lys Ser Ala Leu Arg Asp 500 505 510 Trp Met Gly Cys Pro Ser Gly GlyArg Pro Asp Glu Gly Thr Glu Glu 515 520 525 Glu Thr Glu Val Ile Ile IleGlu Val Asp Glu Glu Gly Gly Gly Ala 530 535 540 Val Ser Ala Ala Ala ValVal Leu Pro Val Leu Leu Leu Leu Leu Val 545 550 555 560 Leu Ala Val GlyLeu Ala Val Phe Phe Phe Arg Arg His Gly Thr Pro 565 570 575 Arg Arg LeuLeu Tyr Cys Gln Arg Ser Leu Leu Asp Lys Val 580 585 590 3 372 PRTArtificial Sequence Description of Artificial Sequence MT1Cat-F 3 MetSer Pro Ala Pro Arg Pro Ser Arg Cys Leu Leu Leu Pro Leu Leu 1 5 10 15Thr Leu Gly Thr Ala Leu Ala Ser Leu Gly Ser Ala Gln Ser Ser Ser 20 25 30Phe Ser Pro Glu Ala Trp Leu Gln Gln Tyr Gly Tyr Leu Pro Pro Gly 35 40 45Asp Leu Arg Thr His Thr Gln Arg Ser Pro Gln Ser Leu Ser Ala Ala 50 55 60Ile Ala Ala Met Gln Lys Phe Tyr Gly Leu Gln Val Thr Gly Lys Ala 65 70 7580 Asp Ala Asp Thr Met Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 85 9095 Asp Lys Phe Gly Ala Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Asp 100105 110 Tyr Lys Asp Asp Asp Asp Lys Tyr Ala Ile Gln Gly Leu Lys Trp Gln115 120 125 His Asn Glu Ile Thr Phe Cys Ile Gln Asn Tyr Thr Pro Lys ValGly 130 135 140 Glu Tyr Ala Thr Tyr Glu Ala Ile Arg Lys Ala Phe Arg ValTrp Glu 145 150 155 160 Ser Ala Thr Pro Leu Arg Phe Arg Glu Val Pro TyrAla Tyr Ile Arg 165 170 175 Glu Gly His Glu Lys Gln Ala Asp Ile Met IlePhe Phe Ala Glu Gly 180 185 190 Phe His Gly Asp Ser Thr Pro Phe Asp GlyGlu Gly Gly Phe Leu Ala 195 200 205 His Ala Tyr Phe Pro Gly Pro Asn IleGly Gly Asp Thr His Phe Asp 210 215 220 Ser Ala Glu Pro Trp Thr Val ArgAsn Glu Asp Leu Asn Gly Asn Asp 225 230 235 240 Ile Phe Leu Val Ala ValHis Glu Leu Gly His Ala Leu Gly Leu Glu 245 250 255 His Ser Ser Asp ProSer Ala Ile Met Ala Pro Phe Tyr Gln Trp Met 260 265 270 Asp Thr Glu AsnPhe Val Leu Pro Asp Asp Asp Arg Arg Gly Ile Gln 275 280 285 Gln Leu TyrGly Gly Glu Ser Gly Phe Pro Thr Lys Met Pro Pro Gln 290 295 300 Pro ArgThr Thr Ser Arg Pro Ser Val Pro Asp Lys Pro Lys Asn Pro 305 310 315 320Thr Tyr Gly Pro Asn Ala Val Ser Ala Ala Ala Val Val Leu Pro Val 325 330335 Leu Leu Leu Leu Leu Val Leu Ala Val Gly Leu Ala Val Phe Phe Phe 340345 350 Arg Arg His Gly Thr Pro Arg Arg Leu Leu Tyr Cys Gln Arg Ser Leu355 360 365 Leu Asp Lys Val 370 4 389 PRT Artificial SequenceDescription of Artificial Sequence MT1PEX-F 4 Met Ser Pro Ala Pro ArgPro Ser Arg Cys Leu Leu Leu Pro Leu Leu 1 5 10 15 Thr Leu Gly Thr AlaLeu Ala Ser Leu Gly Ser Ala Gln Ser Ser Ser 20 25 30 Phe Ser Pro Glu AlaTrp Leu Gln Gln Tyr Gly Tyr Leu Pro Pro Gly 35 40 45 Asp Leu Arg Thr HisThr Gln Arg Ser Pro Gln Ser Leu Ser Ala Ala 50 55 60 Ile Ala Ala Met GlnLys Phe Tyr Gly Leu Gln Val Thr Gly Lys Ala 65 70 75 80 Asp Ala Asp ThrMet Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 85 90 95 Asp Lys Phe GlyAla Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Asp 100 105 110 Tyr Lys AspAsp Asp Asp Lys Thr Tyr Gly Pro Asn Ile Cys Asp Gly 115 120 125 Asn PheAsp Thr Val Ala Met Leu Arg Gly Glu Met Phe Val Phe Lys 130 135 140 LysArg Trp Phe Trp Arg Val Arg Asn Asn Gln Val Met Asp Gly Tyr 145 150 155160 Pro Met Pro Ile Gly Gln Phe Trp Arg Gly Leu Pro Ala Ser Ile Asn 165170 175 Thr Ala Tyr Glu Arg Lys Asp Gly Lys Phe Val Phe Phe Lys Gly Asp180 185 190 Lys His Trp Val Phe Asp Glu Ala Ser Leu Glu Pro Gly Tyr ProLys 195 200 205 His Ile Lys Glu Leu Gly Arg Gly Leu Pro Thr Asp Lys IleAsp Ala 210 215 220 Ala Leu Phe Trp Met Pro Asn Gly Lys Thr Tyr Phe PheArg Gly Asn 225 230 235 240 Lys Tyr Tyr Arg Phe Asn Glu Glu Leu Arg AlaVal Asp Ser Glu Tyr 245 250 255 Pro Lys Asn Ile Lys Val Trp Glu Gly IlePro Glu Ser Pro Arg Gly 260 265 270 Ser Phe Met Gly Ser Asp Glu Val PheThr Tyr Phe Tyr Lys Gly Asn 275 280 285 Lys Tyr Trp Lys Phe Asn Asn GlnLys Leu Lys Val Glu Pro Gly Tyr 290 295 300 Pro Lys Ser Ala Leu Arg AspTrp Met Gly Cys Pro Ser Gly Gly Arg 305 310 315 320 Pro Asp Glu Gly ThrGlu Glu Glu Thr Glu Val Ile Ile Ile Glu Val 325 330 335 Asp Glu Glu GlyGly Gly Ala Val Ser Ala Ala Ala Val Val Leu Pro 340 345 350 Val Leu LeuLeu Leu Leu Val Leu Ala Val Gly Leu Ala Val Phe Phe 355 360 365 Phe ArgArg His Gly Thr Pro Arg Arg Leu Leu Tyr Cys Gln Arg Ser 370 375 380 LeuLeu Asp Lys Val 385 5 391 PRT Artificial Sequence Description ofArtificial Sequence MT4PEX-F 5 Met Ser Pro Ala Pro Arg Pro Ser Arg CysLeu Leu Leu Pro Leu Leu 1 5 10 15 Thr Leu Gly Thr Ala Leu Ala Ser LeuGly Ser Ala Gln Ser Ser Ser 20 25 30 Phe Ser Pro Glu Ala Trp Leu Gln GlnTyr Gly Tyr Leu Pro Pro Gly 35 40 45 Asp Leu Arg Thr His Thr Gln Arg SerPro Gln Ser Leu Ser Ala Ala 50 55 60 Ile Ala Ala Met Gln Lys Phe Tyr GlyLeu Gln Val Thr Gly Lys Ala 65 70 75 80 Asp Ala Asp Thr Met Lys Ala MetArg Arg Pro Arg Cys Gly Val Pro 85 90 95 Asp Lys Phe Gly Ala Glu Ile LysAla Asn Val Arg Arg Lys Arg Asp 100 105 110 Tyr Lys Asp Asp Asp Asp LysThr Tyr Gly Pro Asn Ile Cys Thr Ala 115 120 125 His Phe Asp Ala Val AlaGln Ile Arg Gly Glu Ala Phe Phe Phe Lys 130 135 140 Gly Lys Tyr Phe TrpArg Leu Thr Arg Asp Arg His Leu Val Ser Leu 145 150 155 160 Gln Pro AlaGln Met His Arg Phe Trp Arg Gly Leu Pro Leu His Leu 165 170 175 Asp SerVal Asp Ala Val Tyr Glu Arg Thr Ser Asp His Lys Ile Val 180 185 190 PhePhe Lys Gly Asp Arg Tyr Trp Val Phe Lys Asp Asn Asn Val Glu 195 200 205Glu Gly Tyr Pro Arg Pro Val Ser Asp Phe Ser Leu Pro Pro Gly Gly 210 215220 Ile Asp Ala Val Phe Ser Trp Ala His Asn Asp Arg Thr Tyr Phe Phe 225230 235 240 Lys Asp Gln Leu Tyr Trp Arg Tyr Asp Asp His Thr Arg Arg MetAsp 245 250 255 Pro Gly Tyr Pro Ala Gln Gly Pro Leu Trp Arg Gly Val ProSer Met 260 265 270 Leu Asp Asp Ala Met Arg Trp Ser Asp Gly Ala Ser TyrPhe Phe Arg 275 280 285 Gly Gln Glu Tyr Trp Lys Val Leu Asp Gly Glu LeuGlu Ala Ala Pro 290 295 300 Gly Tyr Pro Gln Ser Thr Ala Arg Asp Trp LeuVal Cys Pro Ser Gly 305 310 315 320 Gly Arg Pro Asp Glu Gly Thr Glu GluGlu Thr Glu Val Ile Ile Ile 325 330 335 Glu Val Asp Glu Glu Gly Gly GlyAla Val Ser Ala Ala Ala Val Val 340 345 350 Leu Pro Val Leu Leu Leu LeuLeu Val Leu Ala Val Gly Leu Ala Val 355 360 365 Phe Phe Phe Arg Arg HisGly Thr Pro Arg Arg Leu Leu Tyr Cys Gln 370 375 380 Arg Ser Leu Leu AspLys Val 385 390 6 425 PRT Artificial Sequence Description of ArtificialSequence MT1EA TM 6 Met Tyr Ala Ile Gln Gly Leu Lys Trp Gln His Asn GluIle Thr Phe 1 5 10 15 Cys Ile Gln Asn Tyr Thr Pro Lys Val Gly Glu TyrAla Thr Tyr Glu 20 25 30 Ala Ile Arg Lys Ala Phe Arg Val Trp Glu Ser AlaThr Pro Leu Arg 35 40 45 Phe Arg Glu Val Pro Tyr Ala Tyr Ile Arg Glu GlyHis Glu Lys Gln 50 55 60 Ala Asp Ile Met Ile Phe Phe Ala Glu Gly Phe HisGly Asp Ser Thr 65 70 75 80 Pro Phe Asp Gly Glu Gly Gly Phe Leu Ala HisAla Tyr Phe Pro Gly 85 90 95 Pro Asn Ile Gly Gly Asp Thr His Phe Asp SerAla Glu Pro Trp Thr 100 105 110 Val Arg Asn Glu Asp Leu Asn Gly Asn AspIle Phe Leu Val Ala Val 115 120 125 His Ala Leu Gly His Ala Leu Gly LeuGlu His Ser Ser Asp Pro Ser 130 135 140 Ala Ile Met Ala Pro Phe Tyr GlnTrp Met Asp Thr Glu Asn Phe Val 145 150 155 160 Leu Pro Asp Asp Asp ArgArg Gly Ile Gln Gln Leu Tyr Gly Gly Glu 165 170 175 Ser Gly Phe Pro ThrLys Met Pro Pro Gln Pro Arg Thr Thr Ser Arg 180 185 190 Pro Ser Val ProAsp Lys Pro Lys Asn Pro Thr Tyr Gly Pro Asn Ile 195 200 205 Cys Asp GlyAsn Phe Asp Thr Val Ala Met Leu Arg Gly Glu Met Phe 210 215 220 Val PheLys Lys Arg Trp Phe Trp Arg Val Arg Asn Asn Gln Val Met 225 230 235 240Asp Gly Tyr Pro Met Pro Ile Gly Gln Phe Trp Arg Gly Leu Pro Ala 245 250255 Ser Ile Asn Thr Ala Tyr Glu Arg Lys Asp Gly Lys Phe Val Phe Phe 260265 270 Lys Gly Asp Lys His Trp Val Phe Asp Glu Ala Ser Leu Glu Pro Gly275 280 285 Tyr Pro Lys His Ile Lys Glu Leu Gly Arg Gly Leu Pro Thr AspLys 290 295 300 Ile Asp Ala Ala Leu Phe Trp Met Pro Asn Gly Lys Thr TyrPhe Phe 305 310 315 320 Arg Gly Asn Lys Tyr Tyr Arg Phe Asn Glu Glu LeuArg Ala Val Asp 325 330 335 Ser Glu Tyr Pro Lys Asn Ile Lys Val Trp GluGly Ile Pro Glu Ser 340 345 350 Pro Arg Gly Ser Phe Met Gly Ser Asp GluVal Phe Thr Tyr Phe Tyr 355 360 365 Lys Gly Asn Lys Tyr Trp Lys Phe AsnAsn Gln Lys Leu Lys Val Glu 370 375 380 Pro Gly Tyr Pro Lys Ser Ala LeuArg Asp Trp Met Gly Cys Pro Ser 385 390 395 400 Gly Gly Arg Pro Asp GluGly Thr Glu Glu Glu Thr Glu Val Ile Ile 405 410 415 Ile Glu Val Asp GluGlu Gly Gly Gly 420 425 7 218 PRT Artificial Sequence Description ofArtificial Sequence MT1PEX 7 Met Cys Asp Gly Asn Phe Asp Thr Val Ala MetLeu Arg Gly Glu Met 1 5 10 15 Phe Val Phe Lys Lys Arg Trp Phe Trp ArgVal Arg Asn Asn Gln Val 20 25 30 Met Asp Gly Tyr Pro Met Pro Ile Gly GlnPhe Trp Arg Gly Leu Pro 35 40 45 Ala Ser Ile Asn Thr Ala Tyr Glu Arg LysAsp Gly Lys Phe Val Phe 50 55 60 Phe Lys Gly Asp Lys His Trp Val Phe AspGlu Ala Ser Leu Glu Pro 65 70 75 80 Gly Tyr Pro Lys His Ile Lys Glu LeuGly Arg Gly Leu Pro Thr Asp 85 90 95 Lys Ile Asp Ala Ala Leu Phe Trp MetPro Asn Gly Lys Thr Tyr Phe 100 105 110 Phe Arg Gly Asn Lys Tyr Tyr ArgPhe Asn Glu Glu Leu Arg Ala Val 115 120 125 Asp Ser Glu Tyr Pro Lys AsnIle Lys Val Trp Glu Gly Ile Pro Glu 130 135 140 Ser Pro Arg Gly Ser PheMet Gly Ser Asp Glu Val Phe Thr Tyr Phe 145 150 155 160 Tyr Lys Gly AsnLys Tyr Trp Lys Phe Asn Asn Gln Lys Leu Lys Val 165 170 175 Glu Pro GlyTyr Pro Lys Ser Ala Leu Arg Asp Trp Met Gly Cys Pro 180 185 190 Ser GlyGly Arg Pro Asp Glu Gly Thr Glu Glu Glu Thr Glu Val Ile 195 200 205 IleIle Glu Val Asp Glu Glu Gly Gly Gly 210 215 8 216 PRT ArtificialSequence Description of Artificial Sequence MT4PEX 8 Met Cys Thr Ala HisPhe Asp Ala Val Ala Gln Ile Arg Gly Glu Ala 1 5 10 15 Phe Phe Phe LysGly Lys Tyr Phe Trp Arg Leu Thr Arg Asp Arg His 20 25 30 Leu Val Ser LeuGln Pro Ala Gln Met His Arg Phe Trp Arg Gly Leu 35 40 45 Pro Leu His LeuAsp Ser Val Asp Ala Val Tyr Glu Arg Thr Ser Asp 50 55 60 His Lys Ile ValPhe Phe Lys Gly Asp Arg Tyr Trp Val Phe Lys Asp 65 70 75 80 Asn Asn ValGlu Glu Gly Tyr Pro Arg Pro Val Ser Asp Phe Ser Leu 85 90 95 Pro Pro GlyGly Ile Asp Ala Val Phe Ser Trp Ala His Asn Asp Arg 100 105 110 Thr TyrPhe Phe Lys Asp Gln Leu Tyr Trp Arg Tyr Asp Asp His Thr 115 120 125 ArgArg Met Asp Pro Gly Tyr Pro Ala Gln Gly Pro Leu Trp Arg Gly 130 135 140Val Pro Ser Met Leu Asp Asp Ala Met Arg Trp Ser Asp Gly Ala Ser 145 150155 160 Tyr Phe Phe Arg Gly Gln Glu Tyr Trp Lys Val Leu Asp Gly Glu Leu165 170 175 Glu Ala Ala Pro Gly Tyr Pro Gln Ser Thr Ala Arg Asp Trp LeuVal 180 185 190 Cys Gly Glu Pro Leu Ala Asp Ala Glu Asp Val Gly Pro GlyPro Gln 195 200 205 Gly Arg Ser Gly Ala Gln Asp Gly 210 215 9 364 PRTArtificial Sequence Description of Artificial Sequence MT1Cat 9 Met SerPro Ala Pro Arg Pro Ser Arg Cys Leu Leu Leu Pro Leu Leu 1 5 10 15 ThrLeu Gly Thr Ala Leu Ala Ser Leu Gly Ser Ala Gln Ser Ser Ser 20 25 30 PheSer Pro Glu Ala Trp Leu Gln Gln Tyr Gly Tyr Leu Pro Pro Gly 35 40 45 AspLeu Arg Thr His Thr Gln Arg Ser Pro Gln Ser Leu Ser Ala Ala 50 55 60 IleAla Ala Met Gln Lys Phe Tyr Gly Leu Gln Val Thr Gly Lys Ala 65 70 75 80Asp Ala Asp Thr Met Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 85 90 95Asp Lys Phe Gly Ala Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Tyr 100 105110 Ala Ile Gln Gly Leu Lys Trp Gln His Asn Glu Ile Thr Phe Cys Ile 115120 125 Gln Asn Tyr Thr Pro Lys Val Gly Glu Tyr Ala Thr Tyr Glu Ala Ile130 135 140 Arg Lys Ala Phe Arg Val Trp Glu Ser Ala Thr Pro Leu Arg PheArg 145 150 155 160 Glu Val Pro Tyr Ala Tyr Ile Arg Glu Gly His Glu LysGln Ala Asp 165 170 175 Ile Met Ile Phe Phe Ala Glu Gly Phe His Gly AspSer Thr Pro Phe 180 185 190 Asp Gly Glu Gly Gly Phe Leu Ala His Ala TyrPhe Pro Gly Pro Asn 195 200 205 Ile Gly Gly Asp Thr His Phe Asp Ser AlaGlu Pro Trp Thr Val Arg 210 215 220 Asn Glu Asp Leu Asn Gly Asn Asp IlePhe Leu Val Ala Val His Glu 225 230 235 240 Leu Gly His Ala Leu Gly LeuGlu His Ser Ser Asp Pro Ser Ala Ile 245 250 255 Met Ala Pro Phe Tyr GlnTrp Met Asp Thr Glu Asn Phe Val Leu Pro 260 265 270 Asp Asp Asp Arg ArgGly Ile Gln Gln Leu Tyr Gly Gly Glu Ser Gly 275 280 285 Phe Pro Thr LysMet Pro Pro Gln Pro Arg Thr Thr Ser Arg Pro Ser 290 295 300 Val Pro AspLys Pro Lys Asn Pro Thr Tyr Gly Pro Asn Ala Val Ser 305 310 315 320 AlaAla Ala Val Val Leu Pro Val Leu Leu Leu Leu Leu Val Leu Ala 325 330 335Val Gly Leu Ala Val Phe Phe Phe Arg Arg His Gly Thr Pro Arg Arg 340 345350 Leu Leu Tyr Cys Gln Arg Ser Leu Leu Asp Lys Val 355 360 10 381 PRTArtificial Sequence Description of Artificial Sequence MT1PEX 10 Met SerPro Ala Pro Arg Pro Ser Arg Cys Leu Leu Leu Pro Leu Leu 1 5 10 15 ThrLeu Gly Thr Ala Leu Ala Ser Leu Gly Ser Ala Gln Ser Ser Ser 20 25 30 PheSer Pro Glu Ala Trp Leu Gln Gln Tyr Gly Tyr Leu Pro Pro Gly 35 40 45 AspLeu Arg Thr His Thr Gln Arg Ser Pro Gln Ser Leu Ser Ala Ala 50 55 60 IleAla Ala Met Gln Lys Phe Tyr Gly Leu Gln Val Thr Gly Lys Ala 65 70 75 80Asp Ala Asp Thr Met Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 85 90 95Asp Lys Phe Gly Ala Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Thr 100 105110 Tyr Gly Pro Asn Ile Cys Asp Gly Asn Phe Asp Thr Val Ala Met Leu 115120 125 Arg Gly Glu Met Phe Val Phe Lys Lys Arg Trp Phe Trp Arg Val Arg130 135 140 Asn Asn Gln Val Met Asp Gly Tyr Pro Met Pro Ile Gly Gln PheTrp 145 150 155 160 Arg Gly Leu Pro Ala Ser Ile Asn Thr Ala Tyr Glu ArgLys Asp Gly 165 170 175 Lys Phe Val Phe Phe Lys Gly Asp Lys His Trp ValPhe Asp Glu Ala 180 185 190 Ser Leu Glu Pro Gly Tyr Pro Lys His Ile LysGlu Leu Gly Arg Gly 195 200 205 Leu Pro Thr Asp Lys Ile Asp Ala Ala LeuPhe Trp Met Pro Asn Gly 210 215 220 Lys Thr Tyr Phe Phe Arg Gly Asn LysTyr Tyr Arg Phe Asn Glu Glu 225 230 235 240 Leu Arg Ala Val Asp Ser GluTyr Pro Lys Asn Ile Lys Val Trp Glu 245 250 255 Gly Ile Pro Glu Ser ProArg Gly Ser Phe Met Gly Ser Asp Glu Val 260 265 270 Phe Thr Tyr Phe TyrLys Gly Asn Lys Tyr Trp Lys Phe Asn Asn Gln 275 280 285 Lys Leu Lys ValGlu Pro Gly Tyr Pro Lys Ser Ala Leu Arg Asp Trp 290 295 300 Met Gly CysPro Ser Gly Gly Arg Pro Asp Glu Gly Thr Glu Glu Glu 305 310 315 320 ThrGlu Val Ile Ile Ile Glu Val Asp Glu Glu Gly Gly Gly Ala Val 325 330 335Ser Ala Ala Ala Val Val Leu Pro Val Leu Leu Leu Leu Leu Val Leu 340 345350 Ala Val Gly Leu Ala Val Phe Phe Phe Arg Arg His Gly Thr Pro Arg 355360 365 Arg Leu Leu Tyr Cys Gln Arg Ser Leu Leu Asp Lys Val 370 375 38011 584 PRT Artificial Sequence Description of Artificial SequenceMT1-MT4PEX 11 Met Ser Pro Ala Pro Arg Pro Ser Arg Cys Leu Leu Leu ProLeu Leu 1 5 10 15 Thr Leu Gly Thr Ala Leu Ala Ser Leu Gly Ser Ala GlnSer Ser Ser 20 25 30 Phe Ser Pro Glu Ala Trp Leu Gln Gln Tyr Gly Tyr LeuPro Pro Gly 35 40 45 Asp Leu Arg Thr His Thr Gln Arg Ser Pro Gln Ser LeuSer Ala Ala 50 55 60 Ile Ala Ala Met Gln Lys Phe Tyr Gly Leu Gln Val ThrGly Lys Ala 65 70 75 80 Asp Ala Asp Thr Met Lys Ala Met Arg Arg Pro ArgCys Gly Val Pro 85 90 95 Asp Lys Phe Gly Ala Glu Ile Lys Ala Asn Val ArgArg Lys Arg Tyr 100 105 110 Ala Ile Gln Gly Leu Lys Trp Gln His Asn GluIle Thr Phe Cys Ile 115 120 125 Gln Asn Tyr Thr Pro Lys Val Gly Glu TyrAla Thr Tyr Glu Ala Ile 130 135 140 Arg Lys Ala Phe Arg Val Trp Glu SerAla Thr Pro Leu Arg Phe Arg 145 150 155 160 Glu Val Pro Tyr Ala Tyr IleArg Glu Gly His Glu Lys Gln Ala Asp 165 170 175 Ile Met Ile Phe Phe AlaGlu Gly Phe His Gly Asp Ser Thr Pro Phe 180 185 190 Asp Gly Glu Gly GlyPhe Leu Ala His Ala Tyr Phe Pro Gly Pro Asn 195 200 205 Ile Gly Gly AspThr His Phe Asp Ser Ala Glu Pro Trp Thr Val Arg 210 215 220 Asn Glu AspLeu Asn Gly Asn Asp Ile Phe Leu Val Ala Val His Glu 225 230 235 240 LeuGly His Ala Leu Gly Leu Glu His Ser Ser Asp Pro Ser Ala Ile 245 250 255Met Ala Pro Phe Tyr Gln Trp Met Asp Thr Glu Asn Phe Val Leu Pro 260 265270 Asp Asp Asp Arg Arg Gly Ile Gln Gln Leu Tyr Gly Gly Glu Ser Gly 275280 285 Phe Pro Thr Lys Met Pro Pro Gln Pro Arg Thr Thr Ser Arg Pro Ser290 295 300 Val Pro Asp Lys Pro Lys Asn Pro Thr Tyr Gly Pro Asn Ile CysThr 305 310 315 320 Ala His Phe Asp Ala Val Ala Gln Ile Arg Gly Glu AlaPhe Phe Phe 325 330 335 Lys Gly Lys Tyr Phe Trp Arg Leu Thr Arg Asp ArgHis Leu Val Ser 340 345 350 Leu Gln Pro Ala Gln Met His Arg Phe Trp ArgGly Leu Pro Leu His 355 360 365 Leu Asp Ser Val Asp Ala Val Tyr Glu ArgThr Ser Asp His Lys Ile 370 375 380 Val Phe Phe Lys Gly Asp Arg Tyr TrpVal Phe Lys Asp Asn Asn Val 385 390 395 400 Glu Glu Gly Tyr Pro Arg ProVal Ser Asp Phe Ser Leu Pro Pro Gly 405 410 415 Gly Ile Asp Ala Val PheSer Trp Ala His Asn Asp Arg Thr Tyr Phe 420 425 430 Phe Lys Asp Gln LeuTyr Trp Arg Tyr Asp Asp His Thr Arg Arg Met 435 440 445 Asp Pro Gly TyrPro Ala Gln Gly Pro Leu Trp Arg Gly Val Pro Ser 450 455 460 Met Leu AspAsp Ala Met Arg Trp Ser Asp Gly Ala Ser Tyr Phe Phe 465 470 475 480 ArgGly Gln Glu Tyr Trp Lys Val Leu Asp Gly Glu Leu Glu Ala Ala 485 490 495Pro Gly Tyr Pro Gln Ser Thr Ala Arg Asp Trp Leu Val Cys Pro Ser 500 505510 Gly Gly Arg Pro Asp Glu Gly Thr Glu Glu Glu Thr Glu Val Ile Ile 515520 525 Ile Glu Val Asp Glu Glu Gly Gly Gly Ala Val Ser Ala Ala Ala Val530 535 540 Val Leu Pro Val Leu Leu Leu Leu Leu Val Leu Ala Val Gly LeuAla 545 550 555 560 Val Phe Phe Phe Arg Arg His Gly Thr Pro Arg Arg LeuLeu Tyr Cys 565 570 575 Gln Arg Ser Leu Leu Asp Lys Val 580 12 592 PRTArtificial Sequence Description of Artificial Sequence MT1-Myc 12 MetSer Pro Ala Pro Arg Pro Ser Arg Cys Leu Leu Leu Pro Leu Leu 1 5 10 15Thr Leu Gly Thr Ala Leu Ala Ser Leu Gly Ser Ala Gln Ser Ser Ser 20 25 30Phe Ser Pro Glu Ala Trp Leu Gln Gln Tyr Gly Tyr Leu Pro Pro Gly 35 40 45Asp Leu Arg Thr His Thr Gln Arg Ser Pro Gln Ser Leu Ser Ala Ala 50 55 60Ile Ala Ala Met Gln Lys Phe Tyr Gly Leu Gln Val Thr Gly Lys Ala 65 70 7580 Asp Ala Asp Thr Met Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 85 9095 Asp Lys Phe Gly Ala Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Glu 100105 110 Gln Lys Leu Ile Ser Glu Glu Asp Leu Tyr Ala Ile Gln Gly Leu Lys115 120 125 Trp Gln His Asn Glu Ile Thr Phe Cys Ile Gln Asn Tyr Thr ProLys 130 135 140 Val Gly Glu Tyr Ala Thr Tyr Glu Ala Ile Arg Lys Ala PheArg Val 145 150 155 160 Trp Glu Ser Ala Thr Pro Leu Arg Phe Arg Glu ValPro Tyr Ala Tyr 165 170 175 Ile Arg Glu Gly His Glu Lys Gln Ala Asp IleMet Ile Phe Phe Ala 180 185 190 Glu Gly Phe His Gly Asp Ser Thr Pro PheAsp Gly Glu Gly Gly Phe 195 200 205 Leu Ala His Ala Tyr Phe Pro Gly ProAsn Ile Gly Gly Asp Thr His 210 215 220 Phe Asp Ser Ala Glu Pro Trp ThrVal Arg Asn Glu Asp Leu Asn Gly 225 230 235 240 Asn Asp Ile Phe Leu ValAla Val His Glu Leu Gly His Ala Leu Gly 245 250 255 Leu Glu His Ser SerAsp Pro Ser Ala Ile Met Ala Pro Phe Tyr Gln 260 265 270 Trp Met Asp ThrGlu Asn Phe Val Leu Pro Asp Asp Asp Arg Arg Gly 275 280 285 Ile Gln GlnLeu Tyr Gly Gly Glu Ser Gly Phe Pro Thr Lys Met Pro 290 295 300 Pro GlnPro Arg Thr Thr Ser Arg Pro Ser Val Pro Asp Lys Pro Lys 305 310 315 320Asn Pro Thr Tyr Gly Pro Asn Ile Cys Asp Gly Asn Phe Asp Thr Val 325 330335 Ala Met Leu Arg Gly Glu Met Phe Val Phe Lys Lys Arg Trp Phe Trp 340345 350 Arg Val Arg Asn Asn Gln Val Met Asp Gly Tyr Pro Met Pro Ile Gly355 360 365 Gln Phe Trp Arg Gly Leu Pro Ala Ser Ile Asn Thr Ala Tyr GluArg 370 375 380 Lys Asp Gly Lys Phe Val Phe Phe Lys Gly Asp Lys His TrpVal Phe 385 390 395 400 Asp Glu Ala Ser Leu Glu Pro Gly Tyr Pro Lys HisIle Lys Glu Leu 405 410 415 Gly Arg Gly Leu Pro Thr Asp Lys Ile Asp AlaAla Leu Phe Trp Met 420 425 430 Pro Asn Gly Lys Thr Tyr Phe Phe Arg GlyAsn Lys Tyr Tyr Arg Phe 435 440 445 Asn Glu Glu Leu Arg Ala Val Asp SerGlu Tyr Pro Lys Asn Ile Lys 450 455 460 Val Trp Glu Gly Ile Pro Glu SerPro Arg Gly Ser Phe Met Gly Ser 465 470 475 480 Asp Glu Val Phe Thr TyrPhe Tyr Lys Gly Asn Lys Tyr Trp Lys Phe 485 490 495 Asn Asn Gln Lys LeuLys Val Glu Pro Gly Tyr Pro Lys Ser Ala Leu 500 505 510 Arg Asp Trp MetGly Cys Pro Ser Gly Gly Arg Pro Asp Glu Gly Thr 515 520 525 Glu Glu GluThr Glu Val Ile Ile Ile Glu Val Asp Glu Glu Gly Gly 530 535 540 Gly AlaVal Ser Ala Ala Ala Val Val Leu Pro Val Leu Leu Leu Leu 545 550 555 560Leu Val Leu Ala Val Gly Leu Ala Val Phe Phe Phe Arg Arg His Gly 565 570575 Thr Pro Arg Arg Leu Leu Tyr Cys Gln Arg Ser Leu Leu Asp Lys Val 580585 590 13 930 PRT Artificial Sequence Description of ArtificialSequence MT1-F/NGFR 13 Met Ser Pro Ala Pro Arg Pro Ser Arg Cys Leu LeuLeu Pro Leu Leu 1 5 10 15 Thr Leu Gly Thr Ala Leu Ala Ser Leu Gly SerAla Gln Ser Ser Ser 20 25 30 Phe Ser Pro Glu Ala Trp Leu Gln Gln Tyr GlyTyr Leu Pro Pro Gly 35 40 45 Asp Leu Arg Thr His Thr Gln Arg Ser Pro GlnSer Leu Ser Ala Ala 50 55 60 Ile Ala Ala Met Gln Lys Phe Tyr Gly Leu GlnVal Thr Gly Lys Ala 65 70 75 80 Asp Ala Asp Thr Met Lys Ala Met Arg ArgPro Arg Cys Gly Val Pro 85 90 95 Asp Lys Phe Gly Ala Glu Ile Lys Ala AsnVal Arg Arg Lys Arg Asp 100 105 110 Tyr Lys Asp Asp Asp Asp Lys Tyr AlaIle Gln Gly Leu Lys Trp Gln 115 120 125 His Asn Glu Ile Thr Phe Cys IleGln Asn Tyr Thr Pro Lys Val Gly 130 135 140 Glu Tyr Ala Thr Tyr Glu AlaIle Arg Lys Ala Phe Arg Val Trp Glu 145 150 155 160 Ser Ala Thr Pro LeuArg Phe Arg Glu Val Pro Tyr Ala Tyr Ile Arg 165 170 175 Glu Gly His GluLys Gln Ala Asp Ile Met Ile Phe Phe Ala Glu Gly 180 185 190 Phe His GlyAsp Ser Thr Pro Phe Asp Gly Glu Gly Gly Phe Leu Ala 195 200 205 His AlaTyr Phe Pro Gly Pro Asn Ile Gly Gly Asp Thr His Phe Asp 210 215 220 SerAla Glu Pro Trp Thr Val Arg Asn Glu Asp Leu Asn Gly Asn Asp 225 230 235240 Ile Phe Leu Val Ala Val His Glu Leu Gly His Ala Leu Gly Leu Glu 245250 255 His Ser Ser Asp Pro Ser Ala Ile Met Ala Pro Phe Tyr Gln Trp Met260 265 270 Asp Thr Glu Asn Phe Val Leu Pro Asp Asp Asp Arg Arg Gly IleGln 275 280 285 Gln Leu Tyr Gly Gly Glu Ser Gly Phe Pro Thr Lys Met ProPro Gln 290 295 300 Pro Arg Thr Thr Ser Arg Pro Ser Val Pro Asp Lys ProLys Asn Pro 305 310 315 320 Thr Tyr Gly Pro Asn Ile Cys Asp Gly Asn PheAsp Thr Val Ala Met 325 330 335 Leu Arg Gly Glu Met Phe Val Phe Lys LysArg Trp Phe Trp Arg Val 340 345 350 Arg Asn Asn Gln Val Met Asp Gly TyrPro Met Pro Ile Gly Gln Phe 355 360 365 Trp Arg Gly Leu Pro Ala Ser IleAsn Thr Ala Tyr Glu Arg Lys Asp 370 375 380 Gly Lys Phe Val Phe Phe LysGly Asp Lys His Trp Val Phe Asp Glu 385 390 395 400 Ala Ser Leu Glu ProGly Tyr Pro Lys His Ile Lys Glu Leu Gly Arg 405 410 415 Gly Leu Pro ThrAsp Lys Ile Asp Ala Ala Leu Phe Trp Met Pro Asn 420 425 430 Gly Lys ThrTyr Phe Phe Arg Gly Asn Lys Tyr Tyr Arg Phe Asn Glu 435 440 445 Glu LeuArg Ala Val Asp Ser Glu Tyr Pro Lys Asn Ile Lys Val Trp 450 455 460 GluGly Ile Pro Glu Ser Pro Arg Gly Ser Phe Met Gly Ser Asp Glu 465 470 475480 Val Phe Thr Tyr Phe Tyr Lys Gly Asn Lys Tyr Trp Lys Phe Asn Asn 485490 495 Gln Lys Leu Lys Val Glu Pro Gly Tyr Pro Lys Ser Ala Leu Arg Asp500 505 510 Trp Met Gly Cys Pro Ser Gly Gly Arg Pro Asp Glu Phe Asn ProGlu 515 520 525 Asp Pro Ile Pro Asp Thr Asn Ser Thr Ser Gly Asp Pro ValGlu Lys 530 535 540 Lys Asp Glu Thr Pro Phe Gly Val Ser Val Ala Val GlyLeu Ala Val 545 550 555 560 Phe Ala Cys Leu Phe Leu Ser Thr Leu Leu LeuVal Leu Asn Lys Cys 565 570 575 Gly Arg Arg Asn Lys Phe Gly Ile Asn ArgPro Ala Val Leu Ala Pro 580 585 590 Glu Asp Gly Leu Ala Met Ser Leu HisPhe Met Thr Leu Gly Gly Ser 595 600 605 Ser Leu Ser Pro Thr Glu Gly LysGly Ser Gly Leu Gln Gly His Ile 610 615 620 Ile Glu Asn Pro Gln Tyr PheSer Asp Ala Cys Val His His Ile Lys 625 630 635 640 Arg Arg Asp Ile ValLeu Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly 645 650 655 Lys Val Phe LeuAla Glu Cys His Asn Leu Leu Pro Glu Gln Asp Lys 660 665 670 Met Leu ValAla Val Lys Ala Leu Lys Glu Ala Ser Glu Ser Ala Arg 675 680 685 Gln AspPhe Gln Arg Glu Ala Glu Leu Leu Thr Met Leu Gln His Gln 690 695 700 HisIle Val Arg Phe Phe Gly Val Cys Thr Glu Gly Arg Pro Leu Leu 705 710 715720 Met Val Phe Glu Tyr Met Arg His Gly Asp Leu Asn Arg Phe Leu Arg 725730 735 Ser His Gly Pro Asp Ala Lys Leu Leu Ala Gly Gly Glu Asp Val Ala740 745 750 Pro Gly Pro Leu Gly Leu Gly Gln Leu Leu Ala Val Ala Ser GlnVal 755 760 765 Ala Ala Gly Met Val Tyr Leu Ala Gly Leu His Phe Val HisArg Asp 770 775 780 Leu Ala Thr Arg Asn Cys Leu Val Gly Gln Gly Leu ValVal Lys Ile 785 790 795 800 Gly Asp Phe Gly Met Ser Arg Asp Ile Tyr SerThr Asp Tyr Tyr Arg 805 810 815 Val Gly Gly Arg Thr Met Leu Pro Ile ArgTrp Met Pro Pro Glu Ser 820 825 830 Ile Leu Tyr Arg Lys Phe Thr Thr GluSer Asp Val Trp Ser Phe Gly 835 840 845 Val Val Leu Trp Glu Ile Phe ThrTyr Gly Lys Gln Pro Trp Tyr Gln 850 855 860 Leu Ser Asn Thr Glu Ala IleAsp Cys Ile Thr Gln Gly Arg Glu Leu 865 870 875 880 Glu Arg Pro Arg AlaCys Pro Pro Glu Val Tyr Ala Ile Met Arg Gly 885 890 895 Cys Trp Gln ArgGlu Pro Gln Gln Arg His Ser Ile Lys Asp Val His 900 905 910 Ala Arg LeuGln Ala Leu Ala Gln Ala Pro Pro Val Tyr Leu Asp Val 915 920 925 Leu Gly930 14 732 PRT Artificial Sequence Description of Artificial SequenceMT1Cat-F/NGFR 14 Met Ser Pro Ala Pro Arg Pro Ser Arg Cys Leu Leu Leu ProLeu Leu 1 5 10 15 Thr Leu Gly Thr Ala Leu Ala Ser Leu Gly Ser Ala GlnSer Ser Ser 20 25 30 Phe Ser Pro Glu Ala Trp Leu Gln Gln Tyr Gly Tyr LeuPro Pro Gly 35 40 45 Asp Leu Arg Thr His Thr Gln Arg Ser Pro Gln Ser LeuSer Ala Ala 50 55 60 Ile Ala Ala Met Gln Lys Phe Tyr Gly Leu Gln Val ThrGly Lys Ala 65 70 75 80 Asp Ala Asp Thr Met Lys Ala Met Arg Arg Pro ArgCys Gly Val Pro 85 90 95 Asp Lys Phe Gly Ala Glu Ile Lys Ala Asn Val ArgArg Lys Arg Asp 100 105 110 Tyr Lys Asp Asp Asp Asp Lys Tyr Ala Ile GlnGly Leu Lys Trp Gln 115 120 125 His Asn Glu Ile Thr Phe Cys Ile Gln AsnTyr Thr Pro Lys Val Gly 130 135 140 Glu Tyr Ala Thr Tyr Glu Ala Ile ArgLys Ala Phe Arg Val Trp Glu 145 150 155 160 Ser Ala Thr Pro Leu Arg PheArg Glu Val Pro Tyr Ala Tyr Ile Arg 165 170 175 Glu Gly His Glu Lys GlnAla Asp Ile Met Ile Phe Phe Ala Glu Gly 180 185 190 Phe His Gly Asp SerThr Pro Phe Asp Gly Glu Gly Gly Phe Leu Ala 195 200 205 His Ala Tyr PhePro Gly Pro Asn Ile Gly Gly Asp Thr His Phe Asp 210 215 220 Ser Ala GluPro Trp Thr Val Arg Asn Glu Asp Leu Asn Gly Asn Asp 225 230 235 240 IlePhe Leu Val Ala Val His Glu Leu Gly His Ala Leu Gly Leu Glu 245 250 255His Ser Ser Asp Pro Ser Ala Ile Met Ala Pro Phe Tyr Gln Trp Met 260 265270 Asp Thr Glu Asn Phe Val Leu Pro Asp Asp Asp Arg Arg Gly Ile Gln 275280 285 Gln Leu Tyr Gly Gly Glu Ser Gly Phe Pro Thr Lys Met Pro Pro Gln290 295 300 Pro Arg Thr Thr Ser Arg Pro Ser Val Pro Asp Lys Pro Lys AsnPro 305 310 315 320 Thr Tyr Gly Pro Asn Glu Phe Asn Pro Glu Asp Pro IlePro Asp Thr 325 330 335 Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys AspGlu Thr Pro Phe 340 345 350 Gly Val Ser Val Ala Val Gly Leu Ala Val PheAla Cys Leu Phe Leu 355 360 365 Ser Thr Leu Leu Leu Val Leu Asn Lys CysGly Arg Arg Asn Lys Phe 370 375 380 Gly Ile Asn Arg Pro Ala Val Leu AlaPro Glu Asp Gly Leu Ala Met 385 390 395 400 Ser Leu His Phe Met Thr LeuGly Gly Ser Ser Leu Ser Pro Thr Glu 405 410 415 Gly Lys Gly Ser Gly LeuGln Gly His Ile Ile Glu Asn Pro Gln Tyr 420 425 430 Phe Ser Asp Ala CysVal His His Ile Lys Arg Arg Asp Ile Val Leu 435 440 445 Lys Trp Glu LeuGly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu 450 455 460 Cys His AsnLeu Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys 465 470 475 480 AlaLeu Lys Glu Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu 485 490 495Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg Phe Phe 500 505510 Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met 515520 525 Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala530 535 540 Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu GlyLeu 545 550 555 560 Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala GlyMet Val Tyr 565 570 575 Leu Ala Gly Leu His Phe Val His Arg Asp Leu AlaThr Arg Asn Cys 580 585 590 Leu Val Gly Gln Gly Leu Val Val Lys Ile GlyAsp Phe Gly Met Ser 595 600 605 Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr ArgVal Gly Gly Arg Thr Met 610 615 620 Leu Pro Ile Arg Trp Met Pro Pro GluSer Ile Leu Tyr Arg Lys Phe 625 630 635 640 Thr Thr Glu Ser Asp Val TrpSer Phe Gly Val Val Leu Trp Glu Ile 645 650 655 Phe Thr Tyr Gly Lys GlnPro Trp Tyr Gln Leu Ser Asn Thr Glu Ala 660 665 670 Ile Asp Cys Ile ThrGln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys 675 680 685 Pro Pro Glu ValTyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro 690 695 700 Gln Gln ArgHis Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu 705 710 715 720 AlaGln Ala Pro Pro Val Tyr Leu Asp Val Leu Gly 725 730 15 729 PRTArtificial Sequence Description of Artificial Sequence MT1PEX-F/NGFR 15Met Ser Pro Ala Pro Arg Pro Ser Arg Cys Leu Leu Leu Pro Leu Leu 1 5 1015 Thr Leu Gly Thr Ala Leu Ala Ser Leu Gly Ser Ala Gln Ser Ser Ser 20 2530 Phe Ser Pro Glu Ala Trp Leu Gln Gln Tyr Gly Tyr Leu Pro Pro Gly 35 4045 Asp Leu Arg Thr His Thr Gln Arg Ser Pro Gln Ser Leu Ser Ala Ala 50 5560 Ile Ala Ala Met Gln Lys Phe Tyr Gly Leu Gln Val Thr Gly Lys Ala 65 7075 80 Asp Ala Asp Thr Met Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 8590 95 Asp Lys Phe Gly Ala Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Asp100 105 110 Tyr Lys Asp Asp Asp Asp Lys Thr Tyr Gly Pro Asn Ile Cys AspGly 115 120 125 Asn Phe Asp Thr Val Ala Met Leu Arg Gly Glu Met Phe ValPhe Lys 130 135 140 Lys Arg Trp Phe Trp Arg Val Arg Asn Asn Gln Val MetAsp Gly Tyr 145 150 155 160 Pro Met Pro Ile Gly Gln Phe Trp Arg Gly LeuPro Ala Ser Ile Asn 165 170 175 Thr Ala Tyr Glu Arg Lys Asp Gly Lys PheVal Phe Phe Lys Gly Asp 180 185 190 Lys His Trp Val Phe Asp Glu Ala SerLeu Glu Pro Gly Tyr Pro Lys 195 200 205 His Ile Lys Glu Leu Gly Arg GlyLeu Pro Thr Asp Lys Ile Asp Ala 210 215 220 Ala Leu Phe Trp Met Pro AsnGly Lys Thr Tyr Phe Phe Arg Gly Asn 225 230 235 240 Lys Tyr Tyr Arg PheAsn Glu Glu Leu Arg Ala Val Asp Ser Glu Tyr 245 250 255 Pro Lys Asn IleLys Val Trp Glu Gly Ile Pro Glu Ser Pro Arg Gly 260 265 270 Ser Phe MetGly Ser Asp Glu Val Phe Thr Tyr Phe Tyr Lys Gly Asn 275 280 285 Lys TyrTrp Lys Phe Asn Asn Gln Lys Leu Lys Val Glu Pro Gly Tyr 290 295 300 ProLys Ser Ala Leu Arg Asp Trp Met Gly Cys Pro Ser Gly Gly Arg 305 310 315320 Pro Asp Glu Phe Asn Pro Glu Asp Pro Ile Pro Asp Thr Asn Ser Thr 325330 335 Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe Gly Val Ser340 345 350 Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu Ser ThrLeu 355 360 365 Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe GlyIle Asn 370 375 380 Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala MetSer Leu His 385 390 395 400 Phe Met Thr Leu Gly Gly Ser Ser Leu Ser ProThr Glu Gly Lys Gly 405 410 415 Ser Gly Leu Gln Gly His Ile Ile Glu AsnPro Gln Tyr Phe Ser Asp 420 425 430 Ala Cys Val His His Ile Lys Arg ArgAsp Ile Val Leu Lys Trp Glu 435 440 445 Leu Gly Glu Gly Ala Phe Gly LysVal Phe Leu Ala Glu Cys His Asn 450 455 460 Leu Leu Pro Glu Gln Asp LysMet Leu Val Ala Val Lys Ala Leu Lys 465 470 475 480 Glu Ala Ser Glu SerAla Arg Gln Asp Phe Gln Arg Glu Ala Glu Leu 485 490 495 Leu Thr Met LeuGln His Gln His Ile Val Arg Phe Phe Gly Val Cys 500 505 510 Thr Glu GlyArg Pro Leu Leu Met Val Phe Glu Tyr Met Arg His Gly 515 520 525 Asp LeuAsn Arg Phe Leu Arg Ser His Gly Pro Asp Ala Lys Leu Leu 530 535 540 AlaGly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu Gly Gln Leu 545 550 555560 Leu Ala Val Ala Ser Gln Val Ala Ala Gly Met Val Tyr Leu Ala Gly 565570 575 Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly580 585 590 Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser Arg AspIle 595 600 605 Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met LeuPro Ile 610 615 620 Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys PheThr Thr Glu 625 630 635 640 Ser Asp Val Trp Ser Phe Gly Val Val Leu TrpGlu Ile Phe Thr Tyr 645 650 655 Gly Lys Gln Pro Trp Tyr Gln Leu Ser AsnThr Glu Ala Ile Asp Cys 660 665 670 Ile Thr Gln Gly Arg Glu Leu Glu ArgPro Arg Ala Cys Pro Pro Glu 675 680 685 Val Tyr Ala Ile Met Arg Gly CysTrp Gln Arg Glu Pro Gln Gln Arg 690 695 700 His Ser Ile Lys Asp Val HisAla Arg Leu Gln Ala Leu Ala Gln Ala 705 710 715 720 Pro Pro Val Tyr LeuAsp Val Leu Gly 725 16 731 PRT Artificial Sequence Description ofArtificial Sequence MT4PEX-F/NGFR 16 Met Ser Pro Ala Pro Arg Pro Ser ArgCys Leu Leu Leu Pro Leu Leu 1 5 10 15 Thr Leu Gly Thr Ala Leu Ala SerLeu Gly Ser Ala Gln Ser Ser Ser 20 25 30 Phe Ser Pro Glu Ala Trp Leu GlnGln Tyr Gly Tyr Leu Pro Pro Gly 35 40 45 Asp Leu Arg Thr His Thr Gln ArgSer Pro Gln Ser Leu Ser Ala Ala 50 55 60 Ile Ala Ala Met Gln Lys Phe TyrGly Leu Gln Val Thr Gly Lys Ala 65 70 75 80 Asp Ala Asp Thr Met Lys AlaMet Arg Arg Pro Arg Cys Gly Val Pro 85 90 95 Asp Lys Phe Gly Ala Glu IleLys Ala Asn Val Arg Arg Lys Arg Asp 100 105 110 Tyr Lys Asp Asp Asp AspLys Thr Tyr Gly Pro Asn Ile Cys Thr Ala 115 120 125 His Phe Asp Ala ValAla Gln Ile Arg Gly Glu Ala Phe Phe Phe Lys 130 135 140 Gly Lys Tyr PheTrp Arg Leu Thr Arg Asp Arg His Leu Val Ser Leu 145 150 155 160 Gln ProAla Gln Met His Arg Phe Trp Arg Gly Leu Pro Leu His Leu 165 170 175 AspSer Val Asp Ala Val Tyr Glu Arg Thr Ser Asp His Lys Ile Val 180 185 190Phe Phe Lys Gly Asp Arg Tyr Trp Val Phe Lys Asp Asn Asn Val Glu 195 200205 Glu Gly Tyr Pro Arg Pro Val Ser Asp Phe Ser Leu Pro Pro Gly Gly 210215 220 Ile Asp Ala Val Phe Ser Trp Ala His Asn Asp Arg Thr Tyr Phe Phe225 230 235 240 Lys Asp Gln Leu Tyr Trp Arg Tyr Asp Asp His Thr Arg ArgMet Asp 245 250 255 Pro Gly Tyr Pro Ala Gln Gly Pro Leu Trp Arg Gly ValPro Ser Met 260 265 270 Leu Asp Asp Ala Met Arg Trp Ser Asp Gly Ala SerTyr Phe Phe Arg 275 280 285 Gly Gln Glu Tyr Trp Lys Val Leu Asp Gly GluLeu Glu Ala Ala Pro 290 295 300 Gly Tyr Pro Gln Ser Thr Ala Arg Asp TrpLeu Val Cys Pro Ser Gly 305 310 315 320 Gly Arg Pro Asp Glu Phe Asn ProGlu Asp Pro Ile Pro Asp Thr Asn 325 330 335 Ser Thr Ser Gly Asp Pro ValGlu Lys Lys Asp Glu Thr Pro Phe Gly 340 345 350 Val Ser Val Ala Val GlyLeu Ala Val Phe Ala Cys Leu Phe Leu Ser 355 360 365 Thr Leu Leu Leu ValLeu Asn Lys Cys Gly Arg Arg Asn Lys Phe Gly 370 375 380 Ile Asn Arg ProAla Val Leu Ala Pro Glu Asp Gly Leu Ala Met Ser 385 390 395 400 Leu HisPhe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu Gly 405 410 415 LysGly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr Phe 420 425 430Ser Asp Ala Cys Val His His Ile Lys Arg Arg Asp Ile Val Leu Lys 435 440445 Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys 450455 460 His Asn Leu Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys Ala465 470 475 480 Leu Lys Glu Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln ArgGlu Ala 485 490 495 Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val ArgPhe Phe Gly 500 505 510 Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val PheGlu Tyr Met Arg 515 520 525 His Gly Asp Leu Asn Arg Phe Leu Arg Ser HisGly Pro Asp Ala Lys 530 535 540 Leu Leu Ala Gly Gly Glu Asp Val Ala ProGly Pro Leu Gly Leu Gly 545 550 555 560 Gln Leu Leu Ala Val Ala Ser GlnVal Ala Ala Gly Met Val Tyr Leu 565 570 575 Ala Gly Leu His Phe Val HisArg Asp Leu Ala Thr Arg Asn Cys Leu 580 585 590 Val Gly Gln Gly Leu ValVal Lys Ile Gly Asp Phe Gly Met Ser Arg 595 600 605 Asp Ile Tyr Ser ThrAsp Tyr Tyr Arg Val Gly Gly Arg Thr Met Leu 610 615 620 Pro Ile Arg TrpMet Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe Thr 625 630 635 640 Thr GluSer Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe 645 650 655 ThrTyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala Ile 660 665 670Asp Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys Pro 675 680685 Pro Glu Val Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro Gln 690695 700 Gln Arg His Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu Ala705 710 715 720 Gln Ala Pro Pro Val Tyr Leu Asp Val Leu Gly 725 730 17578 PRT Artificial Sequence Description of Artificial Sequence MT4-MMP17 Met Gly Arg Arg Pro Arg Gly Pro Gly Ser Pro Arg Gly Pro Gly Pro 1 510 15 Pro Arg Pro Gly Pro Gly Leu Pro Pro Leu Leu Leu Val Leu Ala Leu 2025 30 Ala Ala His Gly Gly Cys Ala Ala Pro Ala Pro Arg Ala Glu Asp Leu 3540 45 Ser Leu Gly Val Glu Trp Leu Ser Arg Phe Gly Tyr Leu Pro Pro Ala 5055 60 Asp Pro Ala Ser Gly Gln Leu Gln Thr Gln Glu Glu Leu Ser Lys Ala 6570 75 80 Ile Thr Ala Met Gln Gln Phe Gly Gly Leu Glu Thr Thr Gly Ile Leu85 90 95 Asp Glu Ala Thr Leu Ala Leu Met Lys Thr Pro Arg Cys Ser Leu Pro100 105 110 Asp Leu Pro Pro Gly Ala Gln Ser Arg Arg Lys Arg Gln Thr ProPro 115 120 125 Pro Thr Lys Trp Ser Lys Arg Asn Leu Ser Trp Arg Val ArgThr Phe 130 135 140 Pro Arg Asp Ser Pro Leu Gly Arg Asp Thr Val Arg AlaLeu Met Tyr 145 150 155 160 Tyr Ala Leu Lys Val Trp Ser Asp Ile Thr ProLeu Asn Phe His Glu 165 170 175 Val Ala Gly Asn Ala Ala Asp Ile Gln IleAsp Phe Ser Lys Ala Asp 180 185 190 His Asn Asp Gly Tyr Pro Phe Asp GlyPro Gly Gly Thr Val Ala His 195 200 205 Ala Phe Phe Pro Gly Asp His HisThr Ala Gly Asp Thr His Phe Asp 210 215 220 Asp Asp Glu Pro Trp Thr PheArg Ser Ser Asp Ala His Gly Met Asp 225 230 235 240 Leu Phe Ala Val AlaVal His Glu Phe Gly His Ala Ile Gly Leu Ser 245 250 255 His Val Ala AlaPro Ser Ser Ile Met Gln Pro Tyr Tyr Gln Gly Pro 260 265 270 Val Gly AspPro Leu Arg Tyr Gly Leu Pro Tyr Glu Asp Arg Val Arg 275 280 285 Val TrpGln Leu Tyr Gly Val Arg Glu Ser Val Ser Pro Thr Ala Gln 290 295 300 LeuAsp Thr Pro Glu Pro Glu Glu Pro Pro Leu Leu Pro Glu Pro Pro 305 310 315320 Asn Asn Arg Ser Ser Thr Pro Pro Gln Lys Asp Val Pro His Arg Cys 325330 335 Thr Ala His Phe Asp Ala Val Ala Gln Ile Arg Gly Glu Ala Phe Phe340 345 350 Phe Lys Gly Lys Tyr Phe Trp Arg Leu Thr Arg Asp Arg His LeuVal 355 360 365 Ser Leu Gln Pro Ala Gln Met His Arg Phe Trp Arg Gly LeuPro Leu 370 375 380 His Leu Asp Ser Val Asp Ala Val Tyr Glu Arg Thr SerAsp His Lys 385 390 395 400 Ile Val Phe Phe Lys Gly Asp Arg Tyr Trp ValPhe Lys Asp Asn Asn 405 410 415 Val Glu Glu Gly Tyr Pro Arg Pro Val SerAsp Phe Ser Leu Pro Pro 420 425 430 Gly Gly Ile Asp Ala Val Phe Ser TrpAla His Asn Asp Arg Thr Tyr 435 440 445 Phe Phe Lys Asp Gln Leu Tyr TrpArg Tyr Asp Asp His Thr Arg Arg 450 455 460 Met Asp Pro Gly Tyr Pro AlaGln Gly Pro Leu Trp Arg Gly Val Pro 465 470 475 480 Ser Met Leu Asp AspAla Met Arg Trp Ser Asp Gly Ala Ser Tyr Phe 485 490 495 Phe Arg Gly GlnGlu Tyr Trp Lys Val Leu Asp Gly Glu Leu Glu Ala 500 505 510 Ala Pro GlyTyr Pro Gln Ser Thr Ala Arg Asp Trp Leu Val Cys Gly 515 520 525 Glu ProLeu Ala Asp Ala Glu Asp Val Gly Pro Gly Pro Gln Gly Arg 530 535 540 SerGly Ala Gln Asp Gly Leu Ala Val Cys Ser Cys Thr Ser Asp Ala 545 550 555560 His Arg Leu Ala Leu Pro Ser Leu Leu Leu Leu Thr Pro Leu Leu Trp 565570 575 Gly Leu 18 790 PRT Artificial Sequence Description of ArtificialSequence NGFR 18 Met Leu Arg Gly Gly Arg Arg Gly Gln Leu Gly Trp His SerTrp Ala 1 5 10 15 Ala Gly Pro Gly Ser Leu Leu Ala Trp Leu Ile Leu AlaSer Ala Gly 20 25 30 Ala Ala Pro Cys Pro Asp Ala Cys Cys Pro His Gly SerSer Gly Leu 35 40 45 Arg Cys Thr Arg Asp Gly Ala Leu Asp Ser Leu His HisLeu Pro Gly 50 55 60 Ala Glu Asn Leu Thr Glu Leu Tyr Ile Glu Asn Gln GlnHis Leu Gln 65 70 75 80 His Leu Glu Leu Arg Asp Leu Arg Gly Leu Gly GluLeu Arg Asn Leu 85 90 95 Thr Ile Val Lys Ser Gly Leu Arg Phe Val Ala ProAsp Ala Phe His 100 105 110 Phe Thr Pro Arg Leu Ser Arg Leu Asn Leu SerPhe Asn Ala Leu Glu 115 120 125 Ser Leu Ser Trp Lys Thr Val Gln Gly LeuSer Leu Gln Glu Leu Val 130 135 140 Leu Ser Gly Asn Pro Leu His Cys SerCys Ala Leu Arg Trp Leu Gln 145 150 155 160 Arg Trp Glu Glu Glu Gly LeuGly Gly Val Pro Glu Gln Lys Leu Gln 165 170 175 Cys His Gly Gln Gly ProLeu Ala His Met Pro Asn Ala Ser Cys Gly 180 185 190 Val Pro Thr Leu LysVal Gln Val Pro Asn Ala Ser Val Asp Val Gly 195 200 205 Asp Asp Val LeuLeu Arg Cys Gln Val Glu Gly Arg Gly Leu Glu Gln 210 215 220 Ala Gly TrpIle Leu Thr Glu Leu Glu Gln Ser Ala Thr Val Met Lys 225 230 235 240 SerGly Gly Leu Pro Ser Leu Gly Leu Thr Leu Ala Asn Val Thr Ser 245 250 255Asp Leu Asn Arg Lys Asn Leu Thr Cys Trp Ala Glu Asn Asp Val Gly 260 265270 Arg Ala Glu Val Ser Val Gln Val Asn Val Ser Phe Pro Ala Ser Val 275280 285 Gln Leu His Thr Ala Val Glu Met His His Trp Ser Ile Pro Phe Ser290 295 300 Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn GlySer 305 310 315 320 Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe LeuGlu Pro Ala 325 330 335 Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg LeuAsn Gln Pro Thr 340 345 350 His Val Asn Asn Gly Asn Tyr Thr Leu Leu AlaAla Asn Pro Phe Gly 355 360 365 Gln Ala Ser Ala Ser Ile Met Ala Ala PheMet Asp Asn Pro Phe Glu 370 375 380 Phe Asn Pro Glu Asp Pro Ile Pro AspThr Asn Ser Thr Ser Gly Asp 385 390 395 400 Pro Val Glu Lys Lys Asp GluThr Pro Phe Gly Val Ser Val Ala Val 405 410 415 Gly Leu Ala Val Phe AlaCys Leu Phe Leu Ser Thr Leu Leu Leu Val 420 425 430 Leu Asn Lys Cys GlyArg Arg Asn Lys Phe Gly Ile Asn Arg Pro Ala 435 440 445 Val Leu Ala ProGlu Asp Gly Leu Ala Met Ser Leu His Phe Met Thr 450 455 460 Leu Gly GlySer Ser Leu Ser Pro Thr Glu Gly Lys Gly Ser Gly Leu 465 470 475 480 GlnGly His Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp Ala Cys Val 485 490 495His His Ile Lys Arg Arg Asp Ile Val Leu Lys Trp Glu Leu Gly Glu 500 505510 Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys His Asn Leu Leu Pro 515520 525 Glu Gln Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Glu Ala Ser530 535 540 Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu Ala Glu Leu Leu ThrMet 545 550 555 560 Leu Gln His Gln His Ile Val Arg Phe Phe Gly Val CysThr Glu Gly 565 570 575 Arg Pro Leu Leu Met Val Phe Glu Tyr Met Arg HisGly Asp Leu Asn 580 585 590 Arg Phe Leu Arg Ser His Gly Pro Asp Ala LysLeu Leu Ala Gly Gly 595 600 605 Glu Asp Val Ala Pro Gly Pro Leu Gly LeuGly Gln Leu Leu Ala Val 610 615 620 Ala Ser Gln Val Ala Ala Gly Met ValTyr Leu Ala Gly Leu His Phe 625 630 635 640 Val His Arg Asp Leu Ala ThrArg Asn Cys Leu Val Gly Gln Gly Leu 645 650 655 Val Val Lys Ile Gly AspPhe Gly Met Ser Arg Asp Ile Tyr Ser Thr 660 665 670 Asp Tyr Tyr Arg ValGly Gly Arg Thr Met Leu Pro Ile Arg Trp Met 675 680 685 Pro Pro Glu SerIle Leu Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val 690 695 700 Trp Ser PheGly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln 705 710 715 720 ProTrp Tyr Gln Leu Ser Asn Thr Glu Ala Ile Asp Cys Ile Thr Gln 725 730 735Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys Pro Pro Glu Val Tyr Ala 740 745750 Ile Met Arg Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg His Ser Ile 755760 765 Lys Asp Val His Ala Arg Leu Gln Ala Leu Ala Gln Ala Pro Pro Val770 775 780 Tyr Leu Asp Val Leu Gly 785 790 19 582 PRT ArtificialSequence Description of Artificial Sequence MT1-MMP 19 Met Ser Pro AlaPro Arg Pro Ser Arg Cys Leu Leu Leu Pro Leu Leu 1 5 10 15 Thr Leu GlyThr Ala Leu Ala Ser Leu Gly Ser Ala Gln Ser Ser Ser 20 25 30 Phe Ser ProGlu Ala Trp Leu Gln Gln Tyr Gly Tyr Leu Pro Pro Gly 35 40 45 Asp Leu ArgThr His Thr Gln Arg Ser Pro Gln Ser Leu Ser Ala Ala 50 55 60 Ile Ala AlaMet Gln Lys Phe Tyr Gly Leu Gln Val Thr Gly Lys Ala 65 70 75 80 Asp AlaAsp Thr Met Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 85 90 95 Asp LysPhe Gly Ala Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Tyr 100 105 110 AlaIle Gln Gly Leu Lys Trp Gln His Asn Glu Ile Thr Phe Cys Ile 115 120 125Gln Asn Tyr Thr Pro Lys Val Gly Glu Tyr Ala Thr Tyr Glu Ala Ile 130 135140 Arg Lys Ala Phe Arg Val Trp Glu Ser Ala Thr Pro Leu Arg Phe Arg 145150 155 160 Glu Val Pro Tyr Ala Tyr Ile Arg Glu Gly His Glu Lys Gln AlaAsp 165 170 175 Ile Met Ile Phe Phe Ala Glu Gly Phe His Gly Asp Ser ThrPro Phe 180 185 190 Asp Gly Glu Gly Gly Phe Leu Ala His Ala Tyr Phe ProGly Pro Asn 195 200 205 Ile Gly Gly Asp Thr His Phe Asp Ser Ala Glu ProTrp Thr Val Arg 210 215 220 Asn Glu Asp Leu Asn Gly Asn Asp Ile Phe LeuVal Ala Val His Glu 225 230 235 240 Leu Gly His Ala Leu Gly Leu Glu HisSer Ser Asp Pro Ser Ala Ile 245 250 255 Met Ala Pro Phe Tyr Gln Trp MetAsp Thr Glu Asn Phe Val Leu Pro 260 265 270 Asp Asp Asp Arg Arg Gly IleGln Gln Leu Tyr Gly Gly Glu Ser Gly 275 280 285 Phe Pro Thr Lys Met ProPro Gln Pro Arg Thr Thr Ser Arg Pro Ser 290 295 300 Val Pro Asp Lys ProLys Asn Pro Thr Tyr Gly Pro Asn Ile Cys Asp 305 310 315 320 Gly Asn PheAsp Thr Val Ala Met Leu Arg Gly Glu Met Phe Val Phe 325 330 335 Lys LysArg Trp Phe Trp Arg Val Arg Asn Asn Gln Val Met Asp Gly 340 345 350 TyrPro Met Pro Ile Gly Gln Phe Trp Arg Gly Leu Pro Ala Ser Ile 355 360 365Asn Thr Ala Tyr Glu Arg Lys Asp Gly Lys Phe Val Phe Phe Lys Gly 370 375380 Asp Lys His Trp Val Phe Asp Glu Ala Ser Leu Glu Pro Gly Tyr Pro 385390 395 400 Lys His Ile Lys Glu Leu Gly Arg Gly Leu Pro Thr Asp Lys IleAsp 405 410 415 Ala Ala Leu Phe Trp Met Pro Asn Gly Lys Thr Tyr Phe PheArg Gly 420 425 430 Asn Lys Tyr Tyr Arg Phe Asn Glu Glu Leu Arg Ala ValAsp Ser Glu 435 440 445 Tyr Pro Lys Asn Ile Lys Val Trp Glu Gly Ile ProGlu Ser Pro Arg 450 455 460 Gly Ser Phe Met Gly Ser Asp Glu Val Phe ThrTyr Phe Tyr Lys Gly 465 470 475 480 Asn Lys Tyr Trp Lys Phe Asn Asn GlnLys Leu Lys Val Glu Pro Gly 485 490 495 Tyr Pro Lys Ser Ala Leu Arg AspTrp Met Gly Cys Pro Ser Gly Gly 500 505 510 Arg Pro Asp Glu Gly Thr GluGlu Glu Thr Glu Val Ile Ile Ile Glu 515 520 525 Val Asp Glu Glu Gly GlyGly Ala Val Ser Ala Ala Ala Val Val Leu 530 535 540 Pro Val Leu Leu LeuLeu Leu Val Leu Ala Val Gly Leu Ala Val Phe 545 550 555 560 Phe Phe ArgArg His Gly Thr Pro Arg Arg Leu Leu Tyr Cys Gln Arg 565 570 575 Ser LeuLeu Asp Lys Val 580 20 10 PRT Artificial Sequence Description ofArtificial Sequence Myc epitope 20 Glu Gln Lys Leu Ile Ser Glu Glu AspLeu 1 5 10

What is claimed is:
 1. A membrane-type 1 matrix metalloproteinase(MT1-MMP) complex formation inhibitor which inhibits the formation of acomplex comprised of plural MT1-MMPs.
 2. A proMMP-2 activation inhibitorwhich inhibits the formation of a complex comprised of plural MT1-MMPs.3. A blocker for cell migration, invasion and/or metastasis whichinhibits the activation of proMMP-2 through inhibiting the formation ofa complex comprised of plural MT1-MMPs.
 4. The blocker according toclaim 3, wherein the cell is a cancer cell.
 5. The inhibitor or blockeraccording to any of claims 1 through 4, wherein the complex comprised ofplural MT1-MMPs is an MT1-MMP homophilic dimer.
 6. The inhibitor orblocker according to any of claims 1 through 5, wherein the complexformation is executed at least with MT1-MMP existing on a cell surface.7. A method for inhibiting MT1-MMP complex formation which comprisesinhibiting the formation of a complex comprised of plural MT1-MMPs.
 8. Amethod for inhibiting the activation of proMMP-2 which comprisesinhibiting the formation of a complex comprised of plural MT1-MMPs.
 9. Amethod for blocking cell migration, invasion and/or metastasis whichcomprises inhibiting the activation of proMMP-2 through inhibiting theformation of a complex comprised of plural MT1-MMPs.
 10. The methodaccording to claim 9, wherein the cell is a cancer cell.
 11. The methodaccording to any of claims 7 through 10, wherein the complex comprisedof plural MT1-MMPs is an MT1-MMP homophilic dimer.
 12. The methodaccording to any of claims 7 through 11, wherein the complex formationis executed at least with MT1-MMP existing on a cell surface.
 13. Asubstance which is active in inhibiting the homophilic dimer formationof MT1-MMP that exists on a cell surface.
 14. The substance according toclaim 13 which is an MT1-MMP hemopexin-like domain (PEX) protein or afragment thereof, or a substance which has substantially equivalentactivity as compared to the same.
 15. The substance according to claim14, wherein the substance is from Cys³¹⁹ to Gly⁵³⁵ of Met plus MT1-MMP,or a protein having substantially equivalent activity as compared to thesame.
 16. A nucleic acid selected from the group consisting of: (i) anucleic acid coding for MT1-MMP PEX; (ii) a nucleic acid coding for afragment of MT1-MMP PEX; (iii) a nucleic acid hybridizable to thenucleic acid as set forth in the above (i); and (iv) a nucleic acidhaving substantially equivalent activity as compared to the nucleic acidas set forth in any of the above (i) through (iii).
 17. The nucleic acidaccording to claim 16, wherein the nucleic acid is a chimeric moleculein which the transmembrane (TM)/cytoplasmic (CP) domains of MT1-MMP aresubstituted with nerve growth factor receptor (NGFR) TM/CP, or amolecule having substantially equivalent activity as compared to thesame.
 18. The nucleic acid according to claim 16, which is a nucleicacid coding for from Cys³¹⁹ to Gly⁵³⁵ of Met plus MT1-MMP, or a nucleicacid having substantially equivalent activity as compared to the same.19. A vector comprising a nucleic acid according to any of claims 16through
 18. 20. A transformed cell comprising (i) a nucleic acidaccording to any of claims 16 through 18 or (ii) a vector according toclaim
 18. 21. The transformed cell according to claim 20 which expressesa chimeric molecule in which MT1-MMP TM/CP is substituted with NGFRTM/CP, or a molecule having substantially equivalent activity ascompared to the same.
 22. A gene therapeutic agent which comprises (i) anucleic acid according to any of claims 16 or 18 or (ii) a vectoraccording to claim
 19. 23. An antibody against (i) an MT1-MMP PEXprotein or a fragment thereof, or (ii) a polypeptide havingsubstantially equivalent activity as compared to the same.
 24. Asubstance capable of binding to MT1-MMP PEX.
 25. The substance accordingto claim 22 which has MT1PEX activity or substantially equivalentactivity as compared to MT1PEX.
 26. A pharmaceutical or veterinary drugcomprising the inhibitor or blocker according to any of claims 1 through6, or the substance, nucleic acid, vector, transformed cell, agent orantibody according to any of claims 13 through 16, 18 through 20, and 22through
 25. 27. The pharmaceutical or veterinary drug according to claim26 for suppressing and/or blocking cell migration, invasion and/ormetastasis.
 28. The pharmaceutical or veterinary drug according to claim26, wherein the cell is a cancer cell.
 29. A screening which comprisesscreening with using, as a marker, the formation of a complex comprisedof plural MT1-MMPs for a substance which inhibits the formation of saidcomplex comprised of said plural MT1-MMPs.
 30. The screening accordingto claim 29, wherein the complex comprised of the plural MT1-MMPs is anMT1-MMP homophilic dimer.
 31. The screening according to claim 29 or 30,wherein a chimeric molecule is used in which MT1-MMP transmembrane(TM)/cytoplasmic (CP) domains are substituted with nerve growth factorreceptor (NGFR) TM/CP.
 32. A substance which (i) inhibits the formationof a complex comprised of plural MT1-MMPs and (ii) is obtained by thescreening according to any of claims 29 through 31.